Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile 

modular platform for functional assays and protein engineering 

 

Thomas Fryer,1,2 Joel David Rogers,1,2 Christopher Mellor,1 Timo N Kohler, 1 Ralph Minter,2,3 

Florian Hollfelder 1,* 

 

 
1Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd, Cambridge 

CB2 1GA, UK. 
2 Antibody Discovery and Protein Engineering, R&D, AstraZeneca, Milstein Building, Granta 

Park, Cambridge, CB21 6GH, UK. 
3 Present address: Alchemab Therapeutics Ltd., 55-56 Russel Square, London, WC1B 4HP, 

UK. 

 

* Corresponding author, email: fh111@cam.ac.uk 

 

  



Abstract 

 

The assembly of robust, modular biological components into complex functional systems is 

central to synthetic biology. Here we apply modular “plug and play” design principles to a 

solid-phase protein display system that facilitates protein purification and functional assays. 

Specifically, we capture proteins on polyacrylamide hydrogel display beads (‘PHD beads’) 

made in microfluidic droplet generators. These monodisperse PHD beads are decorated with 

predefined amounts of anchors: methacrylate-PEG-benzylguanine (BG) and methacrylate-

PEG-chloroalkane (CA), that react covalently with SNAP-/Halo-tag fusion proteins, 

respectively, in a specific, orthogonal and stable fashion. Anchors, and thus proteins, are 

distributed throughout the entire bead volume, allowing attachment of ~109 protein molecules 

per (∅ 20 µm) bead - a higher density than achievable with commercial surface-modified beads. 

We showcase a diverse array of protein modules that enable the secondary capture of proteins, 

either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher 

and SnpTag), in mono- and multivalent display formats. Solid-phase protein binding and 

enzymatic assays are carried out, and incorporating the photocleavable protein PhoCl enables 

the controlled release of modules via visible-light irradiation for functional assays in solution. 

We utilise photocleavage for valency engineering of an anti-TRAIL-R1 scFv, enhancing its 

apoptosis-inducing potency ~50-fold through pentamerisation.      

 

  



Introduction  

 

The analysis of proteins and their use as therapeutics1, enzymes in biocatalysis2 and 

bioremediation3, growth factors for tissue culture4 or targets for binder discovery campaigns5 

is often facilitated by the ability to capture, maintain and manipulate proteins on biocompatible 

surfaces. Protein solid-phase immobilisation is critical to many bioassays (e.g. ELISA6 and 

SPR7 for investigating protein:protein interactions) as it enables washing, modification or 

rebuffering steps and interfaces with robotic workflows, using the protein attachment to handle 

the protein for testing in assays or for direct analyses. Industrial-scale biocatalysis can be 

enhanced by the sequestration/immobilisation of valuable enzymes2 in continuous flow 

biocatalysis,8,9 whilst also offering potential synergistic effects through the co-localisation of 

specific enzymes.10 Proteins immobilised on surfaces have also emerged as useful therapeutic 

agents, enhancing in vivo half-life and providing extra control over drug delivery (both 

temporally and spatially)1,11. Despite the demonstrated utility of immobilised proteins across 

multiple fields, the methods of immobilisation are highly diverse and typically bespoke. Protein 

function and stability can be impacted by surface effects (observed e.g. for immobilised targets 

in phage display5,12 and enzymes in biocatalysis13–15); spectroscopic interference (such as 

autofluorescence16) can negatively affect bioassay sensitivity; the stoichiometry and strength 

of attachment is variable on heterogeneous solid-phase supports; and there can be batch-to-

batch variation that hampers the development of robust and reproducible protocols.  

To simplify engineering of protein capture across a variety of fields, e.g. protein engineering, 

biocatalysis, and therapeutic protein delivery, it is desirable to develop new technologies that 

address many of the aforementioned issues. Ideally, the technology would be versatile, 

controllable and robust (minimising the customisation and optimisation required for each new 

application), the user would have precise control over protein capture density as well as the 

size of the immobilisation matrix, and the capture mechanism and matrix would not themselves 

affect protein functionality or interfere with the envisaged application. To enhance the 

accessibility of such a technology, a core design principle should be that of ‘plug and play’ 

modular components that are easy to produce and engineer. In the context of protein capture 

and manipulation, this should empower a researcher using this system to focus their efforts on 

engineering complex protein-based systems rather than having to extensively validate or 

troubleshoot the individual base components. Robust (i.e. stable, both over time and under 

diverse conditions) protein capture through the trusted modular assembly of “plug and play” 



components thus provides molecular Lego that simplifies the design of e.g. synthetic biology17 

experiments, just as click chemistry18,19 has made aspects of synthetic chemistry generalisable, 

versatile and easy to use. Such robust molecular biology tools have arisen at the interface of 

protein engineering and synthetic biology in recent years, notably: SpyCatcher, amongst 

others20,21, as a plug and play tool for post-translational valency engineering and protein 

purification22; photocontrollable proteins such as PhoCl23 for the spatiotemporal control of 

protein release via light-induced protein backbone cleavage24; or new highly stable and 

versatile protein recognition elements such as the ALFA-tag system25. Whilst extant protein 

immobilisation methods (e.g. Ni-NTA, Streptavidin, Protein A/G, chemical cross-linking) 

have been used successfully with such technologies (e.g. for purification of recombinantly 

expressed versions), no single system incorporates all of the desired traits that are required of 

a system suitable for applications across a wide range of fields: versatility, controllability and 

robustness. We consider that the lack of such a technology curtails the engineerability, and thus 

possible applications, of protein-based systems. 

 

Of the surfaces functionalised with proteins, hydrogels26 are an increasingly important matrix 

for biological applications due to their biocompatibility (permeability, adjustable stiffness, and 

low cytotoxicity). They have found use in single-cell transcriptomics27, mammalian cell 

culture28, in vivo drug delivery devices1 or as artificial cells29. In particular, surface effects can 

be minimised by the absence of a hydrophobic surface that can lead to protein denaturation. 

Hydrogels functionalised with protein have been demonstrated utilising a diverse array of 

capture methods (e.g. anti-His-tag aptamers29, molecular imprinting30, click chemistry24, and 

co-polymerisation with acrylamide31 or through disulphide bond formation32), yet no method 

has been demonstrated that fulfils the criteria of versatility, controllability and robustness. 

 

Here, we introduce a platform that incorporates robust, covalent, site-specific protein capture 

within a hydrogel matrix in a highly modular fashion that offers stability, versatility, and 

accessibility. Using precisely defined, highly specific, orthogonal, and covalent protein capture 

within a hydrogel matrix, a suite of ‘plug and play’ secondary functionality modules were 

developed that achieve non-covalent or covalent capture of defined proteins, at precisely 

defined valencies, and with photocontrollable release of assembled proteins into solution. We 

seek to demonstrate the utility of this platform and associated tools through their application to 

protein binding studies, enzymatic assays, phenotypic cellular assays and therapeutic protein 



engineering. These examples demonstrate the platform’s versatility, as only minimal 

engineering is required to repurpose the system for a new application. 

 

 
Figure 1. Modular polyacrylamide hydrogel display. (a) Monodisperse polyacrylamide hydrogel beads 
are made through the encapsulation of monomers (1: Methacrylate-PEG-benzylguanine (BG), 2: 
Methacrylate-PEG-chloroalkane (CA), 3: Acrylamide, 4: Bis-acrylamide) with polymerisation-
inducing catalysts using droplet-based microfluidics. Upon de-emulsification BG (red) and/or CA 
(blue) are retained within each bead due to co-polymerisation with the hydrogel backbone. (b) Hydrogel 
beads can then be orthogonally functionalised with SNAP- or Halo-tag fusion proteins (red and blue, 
respectively) through covalent reaction with their respective co-polymerised small molecule ligands 
(BG/CA). 

 

Results and Discussion 

 

Design of polyacrylamide hydrogels with titratable protein capture      

Synthesised from components found in most molecular biology labs (e.g. to make  SDS-PAGE 

gels) and with a proven reliability of polymerisation,  polyacrylamide hydrogels are easy to use 

and have readily engineerable mechanical properties (e.g. stiffness and porosity33). Alongside 

their widely known applications as protein separation reagents,  polyacrylamide hydrogels have 

already taken a role as biocompatible scaffolds for the delivery of reagents in microfluidic 

single-cell transcriptomic workflows27, cell culture support matrices (with a particular focus on 

investigating mechanoelastic effects34) and as in vivo drug delivery devices35,36. Despite the 

proven interest in polyacrylamide hydrogels no simple, stable, and modular technology exists 

for their functionalisation with proteins. Polyacrylamide hydrogels consist of chains of 

monomers of acrylamide that are cross-linked by bis-acrylamide in stable polymers. Through 

the variation of acrylamide:bis-acrylamide ratios, hydrogels of different pore sizes and 

a

SNAP

Halo

+

POI

POI

Bead

O

N
H

O O O
O

N
H

N
H S

O

N
H

O O O
O

N
H

N
H S

+
CA

BG
BG BG

CA
CA

b
O

N
H

O O O
O

N
H

N
H S

N
H

O O O O O
O

O

+Bead
CA

BG
BG BG

CA
CA

1

2

3 4



mechanical properties can be brought about as desired for an intended application. Secondary 

properties can also be engineered in, such as dissolution in response to redox, protease or pH 

cues. To enable the capture of proteins, we copolymerised acrylamide and bis-acrylamide 

monomers with methacrylate-modified small molecule ligands (methacrylate-PEG-

benzylguanine (BG) and -chloroalkane (CA); Figure 1a). These ligands act as suicide 

substrates for SNAP-tag37 and Halo-tag38, respectively, and their copolymerisation throughout 

the hydrogel enables completely covalent capture of an array of modular building blocks 

expressed as fusion proteins to these tags (Figure 1b). SNAP-tag and Halo-tag are both well-

established protein tags used across biological fields, and can be expressed in bacterial, yeast 

and mammalian cell lines37. Notably, SNAP-tag and Halo-tag react orthogonally with their 

respective ligands (BG and CA) and have already been used to capture proteins on surfaces39,40, 

yet this orthogonality has not been fully exploited for protein capture on bifunctional surfaces 

and “plug and play” modules for protein engineering and assay design have not been 

developed.  

 

We prepared methacrylate-PEG-benzylguanine/-chloroalkane by reacting methacrylate-NHS 

ester with amine-PEG-benzylguanine/-chloroalkane overnight in a simple click reaction and 

achieved near-quantitative yield (>90 %, as measured by HPLC, Figure S1.1, Table S1.1). The 

products of these reactions can then be directly used for co-polymerisation into polyacrylamide 

hydrogels, and so we subsequently generated BG-functionalised monodisperse beads of 20 µm 

diameter (Ø) using droplet-based microfluidics at ~8 kHz (enabling production of 29 million 

beads per hour). 20 µm beads are readily compatible with downstream analysis technologies 

such as flow cytometry, and represent a readily visualised size for microscopy. However, a 

variety of sizes can be made through the use of different chip geometries and flow rates as the 

particle size is controlled by the size of the microdroplet it is polymerised within, e.g. in the 

InDrop technology (63 µm)27, or in a study by Abate et al. (30 µm)41, and alternative 

technologies can even enable nanometer-scale polyacrylamide particles to be made35,36. Upon 

de-emulsification, BG-functionalised hydrogel beads can be incubated with SNAP-tag fusion 

proteins (such as SNAP-GFP) for covalent capture (Figure 2a). A key feature of 

polyacrylamide hydrogels is their low levels of non-specific interactions with proteins, thus 

enabling the highly specific capture of defined proteins. This specific protein capture is 

exemplified in Figure 2b: only beads functionalised with BG are able to capture SNAP-GFP, 

and there is little-to-no non-specific binding to non-functionalised polyacrylamide beads. PHD 



beads can also be made entirely without the use of microfluidics, by vortexing the aqueous 

monomer solution with surfactant-containing oil (the same compositions as for microfluidics) 

to create polydisperse emulsions. These polydisperse hydrogel beads vary somewhat in size 

but still function as programmed for capture of e.g. SNAP-GFP (Figure S1.2), thus enabling 

their use as protein-capture matrices by researchers without a microfluidic set-up in many of 

the same applications as demonstrated for monodisperse beads within this manuscript.  

 

Next, we sought to quantify the capacity of on-bead coupling. When incubating beads (Ø 20 

µm, 50 µM BG) with increasing amounts of SNAP-GFP, we observed asymptotic saturation 

of the fluorescence signal (after washing of beads) at ~5 x 108 SNAP-GFP molecules per bead, 

and we found this binding behaviour to be highly conserved even when beads are boiled (100oC 

for 10 minutes) before protein capture, demonstrating the high stability of this system (Figure 

2c). In order to estimate the number of molecules required to saturate a bead more accurately, 

we extrapolated the linear part of our saturation curve up to the asymptote. This calculation 

suggests that ~1.5 x 108 SNAP-GFP molecules per bead are bound at saturation (equal, within 

experimental error, to the calculated 1.3 x 108 BG molecules per bead (Ø 20 µm, 50 µM BG 

bead; Figure S1.3a). Such high occupancy levels of immobilised proteins exceed those 

achieved with magnetic beads that bind proteins on their surface by three orders of magnitude42 

(M-280 Streptavidin Dynabeads ~6.6 x 105 IgG molecules per bead Figure S1.3b). The 

difference can be ascribed to the voluminal nature of protein capture, wherein not only is the 

bead’s surface functionalised, but also its interior, as demonstrated by uniform distribution of 

fluorescence in confocal images of the beads (Figure S1.4a, SI). Our confocal data also 

confirmed the long-term stability of the BG-functionalised PHD beads, with an estimated 1/3 

of binding capacity being retained even after 3 years of storage at 4oC (Figure S1.4b, SI). In 

addition to the high levels of protein capture, it is also possible to precisely control the amount 

of captured protein by changing the concentration of BG monomers included in the hydrogel 

polymerisation mix. When the concentration of BG in the initial one-pot pre-polymerisation 

acrylamide mix is varied, the amount of SNAP-GFP captured varies correspondingly; display 

densities spanning at least five orders of magnitude can be brought about at will, and an 

estimated 1.5 x 109 molecules are bound when using 500 uM BG (Figure 2d), demonstrating 

gigavalent capture. The demonstrated stability of the matrix and the ability to precisely control 

protein capture density across multiple orders of magnitude, alongside the previously 



documented ability to modulate the size and mechanical properties of the polyacrylamide 

hydrogel highlight the versatility and precise tunability of the system. 

 

 
Figure 2. Specific, stable and titratable protein capture on polyacrylamide hydrogel beads. (a) BG-

functionalised hydrogel beads are incubated with SNAP-GFP, leading to the covalent capture of SNAP-

GFP on bead. (b) 20 µm PHD beads +/- 50 µM BG were mixed 50:50 and incubated with SNAP-GFP 

followed by washing and imaging (top panel brightfield, bottom panel GFP channel) to detect specific 

GFP attachment. Scalebar: 200 µm. Arrows in the bright-field image indicate non-functionalised beads, 

demonstrating very low non-specific protein binding. (c) 100,000 20 µm, 50 µM BG PHD beads were 

incubated with defined numbers of SNAP-GFP molecules per bead overnight, washed and analysed by 

flow cytometry. The saturation point, i.e. where the addition of extra SNAP-GFP does not lead to an 

increase in on-bead fluorescent signal (dashed line) corresponds to a density of ~150 million attached 

proteins per bead. Black triangles indicate boiled beads, open dashed circles indicate beads handled 

according to our standard procedure (see Methods section).  (d) Five sets of 20 µm PHD beads were 

prepared with the indicated BG loading. All were incubated with an excess of SNAP-GFP, washed, and 

analysed by flow cytometry. The red square highlights the 50 µM BG beads used in (c), that captured 

1.5 x 108 SNAP-GFP molecules per bead, the near-perfect correlation (fitted to a linear model, with an 

intercept at 0 due to background signal subtraction; displayed as a double logarithmic plot to capture 

the wide range of concentrations) between [BG] and green fluorescence shows that a valency range of 

y = 2.1011x
R² = 0.9999

0.005

0.05

0.5

5

50

500

5000

0.05 0.5 5 50 500

No
rm

al
ise

d 
Gr

ee
n 

Fl
uo

re
sc

en
ce

(a
.u

x 
10

3 )

[Benzyl Guanine] (uM) 

1

10

100

1,000

1 10 100 1000No
rm

al
ise

d 
Gr

ee
n 

Fl
uo

re
sc

en
ce

 
(a

.u
x1

03
)

SNAP-GFP molecules per bead (x106)

a

Figure 1: Synthesis and valency control of functionalized polyacrylamide hydrogel beads (a) Methacrylate-PEG-Benzyl 
Guanine

200 um

200 um

c d

+

SNAP GFP

b

Bead

BG
BG BG

BG
BG

BG



105-109 per bead was achieved (for 0.05, 0.5, 5, 50 and 500 µM BG beads respectively). Data are the 

mean of triplicates, normalised to the background signal of PHD beads lacking BG. The error displayed 

is the standard deviation.  
 

Specific protein capture via non-covalent secondary capture modules      

Having established the ability to functionalise polyacrylamide hydrogels for highly stable and 

controllable protein capture, we next sought to establish ‘plug and play’ functional modules 

for protein capture that would enable researchers to use this system in a simple manner, whilst 

taking advantage of the key features of the system (stability, controllability and specificity). 

Whilst we have already demonstrated the direct capture of a protein of interest as a SNAP-tag 

fusion (Figure 2), it is also possible – and greatly enhances the utility of the PHD technology 

as an engineering tool – to use specific secondary capture modules (e.g. affinity reagents fused 

to SNAP-tag) to assemble proteins of interest on bead (Figure 3a). Importantly the use of such 

“plug and play” secondary capture modules enables a researcher to capture specific proteins 

that are not themselves SNAP-tagged, enabling the capture of either non-tagged native proteins 

(e.g. IgG) or recombinantly expressed proteins fused to smaller, less intrusive tags (e.g. SpyTag 

and SnpTag). As the base bead remains the same (20 µm, 50 µM BG), its desired functionality 

can be altered simply by choosing which secondary capture module to initially capture on bead. 

To demonstrate this principle, we fused several secondary capture modules to SNAP-tag for 

immobilisation: SNAP-Protein G for mouse IgG capture (Figure 3b); SNAP-I1943, an anti-

human IgG DARPin, for human IgG capture (Figure 3c); and SNAP-YMB44, an anti-SUMO 

monobody, for capture of SUMO-GFP (Figure 3d). In all figures the specificity of capture is 

demonstrated in control experiments by the lack of fluorescence on beads not functionalised 

with the respective secondary capture module. These secondary capture modules are all readily 

expressed in bacteria, obviating the need to buy expensive affinity reagents for desired 

applications. Further secondary capture modules can be designed based on published sequences 

of affinity reagents or freshly developed through de novo discovery techniques such as phage 

display, and assembled in a modular fashion using e.g. Gibson Assembly (details in Figure 

S1.5, Experimental section S2.3). We demonstrate these protein capture examples not as tasks 

that can uniquely be completed with this system, but as routine applications that could 

subsequently benefit from the associated unique attributes of stability, controllability and 

specificity. 

  



 
Figure 3. Versatile capture of modular building blocks for specific protein capture. (a) BG-
functionalised hydrogel beads can be used to covalently immobilise secondary capture modules (as 
SNAP-tag fusions; SNAP shown in red) that are specific for a desired target protein. (b-d) PHD beads 
(Ø 20 µm; 50 uM BG) +/- their respective modules. (b) SNAP-Protein G, (c) SNAP-I19, or (d) SNAP-
YMB were incubated with their target proteins (Mouse IgG-iFluor 647/blue, human IgG1-AlexaFluor 
488/green, SUMO tag-GFP/bright green, respectively) for 1 hour, washed and analysed by both 
fluorescent microscopy (left-hand panels, beads +/- SNAP-tag capture module were mixed 50:50 and 
imaged together) and flow cytometrically analysed (right-hand panels, beads +/- SNAP-tag capture 
module analysed separately and super-imposed). Scale bars represent 200 µm.  

Modular and orthogonal programming of bead functionality via covalent secondary 

capture modules      

Next, to enable covalent immobilisation of proteins of interest, we designed additional 

secondary capture modules as both SNAP- and Halo-tag fusions to the suite of 

SpyCatcher/SpyTag and SnpCatcher/SnpTag technologies45 (Figure 4a). These protein pairs  

form an isopeptide bond under standard biological reaction conditions and have already been 

applied widely to the modular engineering of proteins (e.g. vaccine design46, protein cyclisation 

for enzyme engineering47, multivalent and multifunctional protein assembly48). In this work we 

use SpyCatcher ΔNC49 (a deimmunised SpyCatcher truncation) and SpyTag00250 (an evolved 

SpyTag with enhanced reaction kinetics). Importantly the two pairs react orthogonally (as do 

Bead

BG
BG BG

BG
BG

BG + +

a

100 101 102 103 104 105 106

RL1-H :: RL1-H

0

50

100

150

200

250
C

ou
nt

Sample Name Median : RL1-H
PG Pos.fcs 195017 
PG Negative.fcs 70.9 

100 101 102 103 104 105 106

BL1-H :: BL1-H

0

200

400

600

800

C
ou

nt

Sample Name Median : BL1-H
SpyCatcher Positive.fcs 314286 
SpyCatcher Negative.fcs 66.0 

100 101 102 103 104 105 106

BL1-H :: BL1-H

0

50

100

150

C
ou

nt

Sample Name Median : BL1-H
YMB Positive Normal Gain.fcs 1063 
YMB Neg Normal Gain.fcs 69.9 

100 101 102 103 104 105 106

BL1-H :: BL1-H

0

200

400

600

C
ou

nt

Sample Name Median : BL1-H
YMB Positive.fcs 227100 
YMB Negative.fcs 7517 

b c

200 um

200 um

200 um

200 um

d

200 um

200 um101 102 103 104

BL1-H :: BL1-H

0

200

400

600

800

C
ou

nt

Sample Name Median : BL1-H
SnpT +.fcs 5652 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SnpC +.fcs 10817 
SnpC -.fcs 51.0 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SpyT +.fcs 5663 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

100

200

300

400

C
ou

nt

Sample Name Median : BL1-H
I19 +.fcs 5222 
I19 -.fcs 52.0 

Cy5 fluorescence Green fluorescence

Green fluorescence

- SNAP-Protein G
+ SNAP-Protein G

- SNAP-I19
+ SNAP-I19

- SNAP-YMB
+ SNAP-YMB



SNAP-tag and Halo-tag) enabling the specific modular construction of multifunctional beads 

with relative ease. Whilst SpyTag and SnpTag have both been incorporated into hydrogel 

frameworks previously (PEG-functionalised31 or all-protein hydrogels51) the versatility of these 

systems is limited compared to that displayed here in which any and all arrangements of protein 

pairs can be assembled on bead (Figure 4 b-e) simply by exchanging the covalent secondary 

capture module first captured on bead. Due to the orthogonality of the four protein capture 

technologies employed (SNAP-tag, Halo-tag, SpyCatcher, SnpCatcher), specific capture of 

target proteins can be programmed by simply functionalising beads +/- any desired component. 

In Figure 4f we demonstrate the highly controlled capture of GFP-SnpT/mCherry-SpyT based 

solely upon the previous functionalisation of beads with/without SNAP-SpyCatcher/Halo-

SnpCatcher. These beads now exhibit programmed bifunctionality (both GFP and mCherry 

fluorescence), and serve to demonstrate the versatility, modularity and orthogonality of the 

PHD technology. As before, researchers can design and express further capture modules and 

functionalities with relative ease through the use of modular Gibson Assembly.  



 

a

+

Covalent capture 
modules

Bead
CA

BG
BG BG

CA
CA

SNAP SpyC

Fluorescent conjugate 
modules

GFP+ SpyT

100 101 102 103 104 105 106

RL1-H :: RL1-H

0

50

100

150

200

250

C
ou

nt

Sample Name Median : RL1-H
PG Pos.fcs 195017 
PG Negative.fcs 70.9 

100 101 102 103 104 105 106

BL1-H :: BL1-H

0

200

400

600

800

C
ou

nt

Sample Name Median : BL1-H
SpyCatcher Positive.fcs 314286 
SpyCatcher Negative.fcs 66.0 

b c

200 µm

101 102 103 104

BL1-H :: BL1-H

0

200

400

600

800

C
ou

nt

Sample Name Median : BL1-H
SnpT +.fcs 5652 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SnpC +.fcs 10817 
SnpC -.fcs 51.0 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SpyT +.fcs 5663 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

100

200

300

400

C
ou

nt

Sample Name Median : BL1-H
I19 +.fcs 5222 
I19 -.fcs 52.0 

- SNAP-SpyC
+ SNAP-SpyC

- SNAP-SpyT
+ SNAP-SpyT

Green fluorescence Green fluorescence

200 µm

200 µm

200 µm

d e
101 102 103 104

BL1-H :: BL1-H

0

200

400

600

800

C
ou

nt

Sample Name Median : BL1-H
SnpT +.fcs 5652 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SnpC +.fcs 10817 
SnpC -.fcs 51.0 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SpyT +.fcs 5663 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

100

200

300

400

C
ou

nt

Sample Name Median : BL1-H
I19 +.fcs 5222 
I19 -.fcs 52.0 

101 102 103 104

BL1-H :: BL1-H

0

200

400

600

800

C
ou

nt

Sample Name Median : BL1-H
SnpT +.fcs 5652 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SnpC +.fcs 10817 
SnpC -.fcs 51.0 

101 102 103 104

BL1-H :: BL1-H

0

500

1.0K

1.5K

2.0K

C
ou

nt

Sample Name Median : BL1-H
SpyT +.fcs 5663 
SnpT -.fcs 71.9 

101 102 103 104

BL1-H :: BL1-H

0

100

200

300

400

C
ou

nt

Sample Name Median : BL1-H
I19 +.fcs 5222 
I19 -.fcs 52.0 

- SNAP-SnpC
+ SNAP-SnpC

- SNAP-SnpT
+ SNAP-SnpT

Green fluorescence Green fluorescence

200 µm

200 µm200 µm

200 µm

0 103

BluFL1

0

-10 2

102

103

104

105

YG
FL

2

SampleID Median : BluFL1 Median : YGFL2
HaloSnpCatcher SnapSpyCatcher 721 37538 
Snap-SpyCatcher 6.89 32650 
Halo-SnpCatcher 766 -13.2 
Negative 15.9 -12.1 

SNAP-
SpyCatcher - - + +

Halo-
SnpCatcher - + + -

f

Green fluorescence

m
Ch

er
ry

flu
or

es
ce

nc
e

f



Figure 4 Versatile, orthogonal and covalent capture of target proteins. (a) BG/CA-functionalised 
hydrogel beads can be used to covalently immobilise secondary covalent capture modules (as SNAP-
tag or Halo-tag fusions) that specifically react with their partner tag. Throughout the figure, relevant 
protein domains are indicated as: SNAP-tag (red circle), Halo-tag (blue circle), SpyCatcher (orange 
pac-man), SnpCatcher (blue pac-man), SpyTag (green diamond), SnpTag (pink diamond), GFP (green 
circle), mCherry (purple circle). (b-e) Monofunctionalised PHD beads (50 µM BG, Ø 20 µm) were 
incubated +/- SNAP fusion proteins: (b) SNAP-SpyCatcher; (c) SNAP-SpyTag; (d) SNAP-SnpCatcher; 
or (e) SNAP-SnpTag. These beads were then mixed and incubated with their respective target proteins 
(b: GFP-SpyTag; c: GFP-SpyCatcher; d: GFP-SnpTag; e: GFP-SnpCatcher) for 1 hour, washed and 
analysed by both fluorescent microscopy (left-hand panels) and flow cytometry (right-hand panels). 
Scale bars represent 200 µm. (f) Bifunctionalised PHD beads (50 µM BG, 50 µM CA, Ø 20 µm) were 
incubated +/- SNAP-SpyCatcher and/or Halo-SnpCatcher; these beads were subsequently incubated 
with both GFP-SnpTag and mCherry-SpyTag for 1 hour, washed and analysed by flow cytometry. 

 

Application of PHD beads to bioassays: protein-protein interactions, enzymatic catalysis 

and bacteriolysis 

Due to the modularity and robustness of the PHD technology it is facile to design and 

implement bioassays. We demonstrate this for assaying protein:protein interactions – an 

extremely common bioassay which is key to understanding basic molecular interactions (e.g. 

in the development of protein-based therapeutics) – by carrying out an investigation into the 

binding affinity of SNAP-Protein G for a mouse IgG subtype (Figure 5a). We incubated 

SNAP-Protein G functionalised beads with a titration series of fluorescently labelled mouse 

IgG2b, before washing away unbound IgG and measuring the amount of binding by flow 

cytometry. This experiment bears close resemblance to those designed for yeast surface 

display-mediated measurements of binding affinity52 that have proved to compare favourably 

to the “gold-standard” method of surface plasmon resonance. An affinity of 30.1 nM was 

calculated, in close accordance with published data53 on Protein G binding to mouse IgG (41.5 

nM, we note that data is binding to total mouse IgG rather than an individual subclass).  

 

Having demonstrated the utility of PHD beads for assaying protein:protein interactions, we 

wished to also highlight their suitability for simplifying and improving the quality of high-

throughput assays. SNAP-SnpTag was captured directly from bacterial cell lysate and probed 

by subsequent incubation with GFP-SnpCatcher. A minimal volume of 2x concentrated cell 

lysate (1 µl) corresponding to ~2 µl of culture volume (1/500 of the largest volume tested) was 

found to already saturate 50,000 20 µm, 50 µM BG beads (Figure S1.6). Direct capture of a 

protein of interest from cell lysate obviates the need for a separate purification step, whilst the 

precise control over protein capture through user-controlled BG concentration and bead 



number effectively achieves expression level normalisation for a subsequent assay. Protein 

expression, lysis, on-bead capture and the subsequent assay (flow cytometry) were all carried 

out in a 96 deep-well plate format; combining the PHD beads with high-throughput, sensitive 

techniques such as flow cytometry creates a powerful platform with which multiple parameters 

(e.g. affinity and specificity) can be examined simultaneously, and assays can be multiplexed 

for even greater throughput54. 

 

In addition to protein:protein interactions another common form of bioassay is enzymatic 

catalysis, in which the accumulation of product or loss of substrate is followed over time. The 

immobilisation of enzymes is of great interest for industrial biocatalysis2 and can also serve to 

provide a simple method of delivering a defined concentration of protein to a given assay – an 

important feature when comparing the activity of enzyme variants in a directed evolution 

experiment for instance. To demonstrate the precise control of enzyme concentration for use 

in a subsequent bioassay we captured P9155-SpyTag, a phosphotriesterase, on SNAP-

SpyCatcher-functionalised beads. The number of beads per reaction was varied, and the 

accumulation of product followed by an increase in fluorescence signal (Figure 5b). A near-

perfect linear relationship is seen between bead number per reaction and catalytic activity, 

highlighting two key points: firstly that the captured enzyme remains functional on bead, and 

secondly that the quantity of enzyme delivered to an assay can be precisely controlled by the 

number of enzyme-functionalised beads delivered to that assay. In addition, as a proof-of-

principle, this experiment also highlights the compatibility of PHD beads with the cell-free 

expression of proteins, as P91-SpyTag was expressed using PURExpress and directly captured 

on-bead from the in vitro expression reaction. Cell-free expression of proteins is now a well-

established field56 with commercial products available, and can enable the rapid, (ultra)high-

throughput expression of even cytotoxic proteins57. 

 

Next, to demonstrate that our platform’s applications are not limited to cell-free bioassays, we 

designed a microtitre plate- and flow cytometry-compatible sensor for bacteriolysis to facilitate 

the discovery of antibacterials (Figure 5c). PHD beads were first functionalised with the 

SNAP-SpyCatcher covalent capture module before being incubated with E. coli which 

expressed GFP-SpyTag intracellularly and had been exposed to carbenicillin at a range of 

different concentrations (0-500 µg/mL) and under three different conditions: static culture; 

culture diluted 1:1 in PBS; and culture resuspended in fresh media (Figure 5d). Bacteriolysis 



is sensed by the release of GFP-SpyTag from lysed bacteria and its subsequent capture on 

SNAP-SpyC functionalised PHD beads. These sensor beads can then be recovered and 

quantitatively analysed by flow cytometry. We observed that resuspension of cells in fresh 

media was necessary for the maximal induction of bacteriolysis, and we further note that these 

results implicate carbenicillin (and/or related molecules) as an effective protein extraction 

reagent. The observation that resuspension is required for cell lysis is supported by the 

literature58, and a mechanistic understanding of how carbenicillin acts59 (as an inhibitor of 

transpeptidases required for cell wall biosynthesis). 

 

 
Figure 5. PHD beads in designed functional bioassays. (a) PHD beads (50 µM BG, Ø 20 µm) 
functionalised with/without SNAP-Protein G were incubated with a titration series of fluorescently 
labelled mouse IgG2b at room temperature with rolling for the indicated times. Beads were recovered, 
washed, and analysed by flow cytometry. Data are the mean of triplicates; and the curve was fitted to 
the equation Fluorescence = (FluorescenceMax x [IgG])/(KD + [IgG]).  (b) In vitro-expressed P91-
SpyTag was captured on bead, washed and incubated with 50 µM substrate (fluoresceine-
di(diethylphosphate)) in 100 µl volume. Bead number per well was varied as indicated. The initial 90 
minutes of reaction was used to calculate the catalytic activity. Data is presented normalised to non-
functionalised beads to control for background hydrolysis of the substrate. (c) Overview of bacteriolysis 
sensor design. PHD beads functionalised with the SNAP-SpyC covalent capture module are incubated 
with bacterial cells expressing GFP-SpyT. Only upon lysis will the GFP-SpyT be released into solution 
and be able to be captured on the sensor beads. (d) E. coli cells expressing GFP-SpyTag were grown 
overnight with induction of protein expression. Static cultures (blue), cultures resuspended in fresh 

0

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culture media (orange), and static cultures diluted 1:1 with PBS (grey) were incubated with a range of 
carbenicillin concentrations for 90 minutes at 37 oC in triplicate. Cultures were pelleted and the 
supernatant transferred to incubate with SpyCatcher-functionalised PHD beads for 60 minutes. Beads 
were washed twice and then analysed by flow cytometry. 
 

 

Valency engineering and photocontrolled release of antibody drugs for phenotypic assays 

As an extension to the tools already exhibited, we sought to develop a method of releasing 

captured proteins into solution upon exposure to a specific cue. Ideally this process would be 

simple, highly controllable and stable, without the requirement for addition of further reagents. 

Recent advances have enabled the use of genetically encoded photocontrollable elements for 

micropatterning24 and control of hydrogel stiffness60 utilising the photocleavable protein 

PhoCl23. Upon exposure to violet light (405 nm), PhoCl cleaves its own backbone, thus 

allowing for the controlled release of attached proteins. Previous attempts to use PhoCl for the 

controlled release of proteins from hydrogels used click chemistry for immobilisation, which 

can negatively affect protein functionality through non-site-specific protein capture as well as 

limiting the engineerability of the system through a lack of orthogonality and easy modularity24. 

Therefore, we designed and tested a new modular building block, SNAP-PhoCl-SpyCatcher, 

that would release the SpyCatcher and any associated cargo from the hydrogel (Figure 6a). 

Cleavage in solution was first verified, with significant cleavage seen after just one minute of 

exposure to light (Figure 6b). Due to the transparent nature of the PHD beads, we expected 

photocleavage to retain comparable efficiency when the SNAP-PhoCl-SpyCatcher modular 

building block is captured on bead. To test this, beads were functionalised with SNAP-PhoCl-

SpyCatcher and exposed to 405 nm light. After light exposure (to prevent any effect of 

photobleaching) beads were incubated with mCherry-SpyTag to assay for PhoCl cleavage, and 

hence loss of the SpyCatcher entity from bead. A decrease in mCherry fluorescence thus 

indicates a release of SpyCatcher from the bead, and after 15 minutes of exposure to 405 nm 

light around 80% of protein is released (36% decrease in fluorescence after 5 minutes, and 79% 

after 15 minutes; Figure 6c). Improved photocleavage proteins, such as the recently developed 

PhoCl2,61 can be easily incorporated based on the modular design.  

 

Many protein:protein interactions rely upon specific valencies of the interacting partners to 

trigger a specific cellular response62,63. Engineering the valency state of protein-based 

therapeutics that are designed to drug such biological systems typically relies upon laborious 

in-frame cloning and expression, limiting the capacity of a researcher to investigate many 



different drugs at many different valencies. The SpyCatcher technology has already been 

demonstrated to facilitate valency engineering through the post-translational assembly of 

monomeric nanobody-SpyTag into multivalent constructs via capture on SpyCatcher-coiled 

coil domain fusions22. We build upon this work by capturing SpyTag fusion proteins on PHD 

beads functionalised for valency engineering, thus taking advantage of surface immobilisation 

for washing and handling, and the subsequent release of assay components (e.g. in response to 

a supplied cue of light) to remove surface effects completely. To this end, we mounted distinct 

populations of beads with one of six SNAP-PhoCl-SpyCatcher fusion proteins (SNAP-PhoCl-

SpyCatcher1-6, differing in the number of SpyCatcher repeats). Subsequent incubation with a 

monomeric SpyTag fusion protein results in assembly into photoreleasable, tunably 

multivalent constructs, depending only on the SpyCatcher module used (Figure 6a). An anti-

TRAIL-R1 scFv64 (3B04) was chosen as a candidate for molecular engineering as related scFv 

TRAIL-R1 agonists65 reformatted as IgG had undergone clinical trial, with no clinical benefit 

seen in either non-small-cell lung cancer66 or colorectal cancer67. TRAIL-R1 is widely 

considered to signal as a trimer and, in vivo, is agonised by the trimeric TRAIL68, and we 

therefore hypothesised that enhanced potency could be achieved by engineering multivalent 

versions of the scFv. Similar multivalency engineering approaches have been carried out for 

nanobodies that target TRAIL-R2, a highly related receptor also found to be overexpressed on 

cancer cells, with great success22,69, but to our knowledge no such investigation has been carried 

out for scFvs targeting TRAIL-R1.  

 

Initially, we investigated the effect of making 3B04 trivalent (Figure 6d) through the 

incubation of 3B04-SpyTag with beads functionalised with SNAP-PhoCl-SpyCatcher3 and the 

subsequent exposure of half of these beads to 405 nm light. We observed that the trivalent 

engineered scFv construct is more potent than the monovalent scFv, and also that release of 

the multivalent assembly from the bead surface is necessary to fully induce apoptosis. The 

lower potency of the on-bead trivalent scFv is likely due to the sequestration of trivalent scFv 

assemblies within the volume of the bead, inaccessible to the cell surface receptors. This cell 

exclusion effect is also noted in a study by Abate et al.70, in which yeast cells encapsulated 

within a microdroplet with a polyacrylamide hydrogel bead only grow in the peripheral 

aqueous zone between bead and droplet edge. It was straightforward to further engineer the 

valency state of 3B04-SpyT through incubation with separate bead populations, each 

functionalised with one of the six valency engineering modules (SNAP-PhoCl-SpyCatcher1-6). 



Subsequent exposure to 405 nm light released each of the fully 3B04-conjugated valency 

engineering modules into solution with little-to-no under-functionalised modules being 

observed in SDS-PAGE gel analysis (Figure 6e). We incubated serial dilutions of each of these 

constructs with HeLa cells for 2 hours and measured apoptosis induction using a fluorogenic 

caspase-3 substrate (NucView 488; Figure 6f). Importantly the data are presented normalised 

to the scFv concentration in the assay (measured by the A280 value of the assembled construct 

and multiplied by the number of scFv molecules captured on an assembly), therefore the 

observed shift in potency is      due to the effect of different valencies rather than a concentration 

effect. Decreases in the EC50 values indicate significant increases in potency for all multivalent 

constructs over monovalent scFv (e.g. >50-fold for the pentavalent versus monovalent format; 

Table 1). Intriguingly, we observe an approximately two-fold reduction in potency when 

increasing scFv valency from 4x or 5x to 6x. This notion is consistent with previous 

observations that TRAIL-R1 signalling is dependent not only on trimerisation, but also on co-

localisation of numerous TRAIL-R1 trimers within lipid rafts71. We speculate that the 4x and 

5x constructs may promote trimer formation whilst also forming a lateral ‘bridge’ between 

consecutive TRAIL-R1 trimers, whereas the 6x construct may only enhance formation of a pair 

of trimers.  

 

 

 



 
Figure 6: Photocontrolled valency engineering for antibody drug phenotypic assays. (a) Beads can be 
functionalised with the valency engineering covalent capture modules (SNAP-PhoCl-SpyC1-6) and 
subsequently used to capture SpyT-POI (here scFv-SpyT). Upon exposure to 405 nm light the PhoCl 
protein self-cleaves and releases the valency-modified assembly into solution. (b) SNAP-PhoCl-SpyC1 
was exposed to 405 nm light for the indicated durations and the samples loaded on a denaturing SDS-
PAGE gel for analysis of cleavage. (c) PHD beads functionalised with SNAP-PhoCl-SpyC1 were 
exposed to 405 nm light for the indicated durations. Beads were then washed and incubated with 
mCherry-SpyTag followed by flow cytometry. Data represent the mean of triplicates, and the 
fluorescence values are displayed above each bar. (d) 100,000 20 µm 50 µM BG beads for each sample 
were incubated with SNAP-PhoCl-SpyC3 and then 3B04-SpyTag. Samples were then treated +/- light 
and incubated with HeLa cells to measure apoptosis induction. (e) Beads functionalised with each of 

hv

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the indicated SNAP-PhoCl-SpyC1-6 valency engineering covalent capture modules were subsequently 
functionalised with scFv-SpyTag, washed, and exposed to 405 nm light for 10 minutes. Samples were 
centrifuged, and 9 µL of the supernatant loaded on a denaturing SDS-PAGE gel. (f) Released 
multivalent assemblies from (e) were incubated with HeLa cells for 2 hours at the indicated 
concentrations. Cells were then assayed for apoptosis induction by incubation with NucView 488 and 
subsequent flow cytometry. Effective scFv concentration is the concentration of scFv in each well 
regardless of its multivalent state; data were obtained in triplicate; the dashed line indicates 50 % 
apoptosis; and the sigmoid curves are fitted Hill equations. 

 

Table 1 EC50 values and standard deviations for multivalent antibody-induced cancer cell apoptosis. 
All EC50 values differ significantly from each other (p < 0.005, Welch’s two-tailed t-test). Conditions 
as per Figure 6f.  

 

 

 

 

 

 

 

 

 

Conclusions and Implications 

 

An accessible, personalised technology platform for protein immobilisation       

In contrast to commercial microbeads (made of e.g. polystyrene), PHD beads have user-

definable attachment points and therefore bring customisable orthogonality and control over 

the valency of protein immobilisation into the hands of the researcher, who can exert this 

control in their laboratory simply by modifying the concentration of components in the 

hydrogel synthesis mixture. This: reduces reliance on commercial suppliers; avoids batch-to-

batch variation outside the control of the researcher; enables a simple method for delivering 

user-defined amounts of protein to bioassays; and allows personalised variation of the type of 

tags used. Furthermore, the simple microfluidic bead synthesis ensures monodispersity at a 

level of control that is not available for commercial beads, providing flexibility and robustness 

to bioassays.  Attachment points are selective (allowing e.g. direct purification of the protein 

from a cell lysate), which is brought about by covalent tagging. In addition to SNAP- and Halo-

tag as used in this study, other tags are also available which could further expand the 

scFv valency EC50 (nM) 

1 114 ± 18 

2 15.8 ± 1.2 

3 8.21 ± 0.26 

4 2.39 ± 0.17 

5 1.99 ± 0.17 

6 5.88 ± 0.52 

 



orthogonality and engineerability of this system72,73. The site-specific nature of protein capture 

minimises the potential impact of immobilisation on the activity of the protein of interest, 

whilst the covalent nature ensures that captured proteins remain stably associated with the 

hydrogel and do not leach into solution. Surface effects that are frequently encountered when 

proteins are physically immobilised on plastic surfaces are minimized, and hydrogels can be 

expected to mimic the natural environment for soluble proteins much better than a hydrophobic 

surface. The 3D distribution of attachment points throughout the hydrogel volume (rather than 

the surface of commercial microbeads) enables each bead to be decorated with 150 million 

protein molecules or more (~1.5 billion for 500 µM BG beads) in contrast with ~660 thousand 

protein molecules captured on commercial streptavidin beads (Figure SI3b). Finally, hydrogel 

beads are optically transparent, so that fluorescent measurements are possible and strong signal 

over background can be detected in all fluorescent channels, while commercial magnetic 

polystyrene beads exhibit autofluorescence in relevant channels, limiting assay sensitivity16. 

 

 

Versatile Assay Formatting      

Based on the modular design principles of synthetic biology, PHD beads can be decorated by 

attaching tagged protein constructs in a generic way, in an effectively “plug and play” solution 

for biological experiments and engineering. This approach mirrors ‘click chemistry’18 by 

providing universal procedures for attachment that do not have to be adjusted on a case-by-

case basis. Direct capture of POIs as SNAP or Halo-tag constructs initially simplifies protein 

purification directly from cell lysates, and this direct capture can be further augmented by the 

use of secondary capture modules which enable the expansion of protein capture to endogenous 

untagged targets (e.g. IgG) through the use of defined recombinant affinity reagents. We have 

developed a suite of these, focussing on bacterially expressible scaffolds to increase 

accessibility to the technology, and this suite could be readily expanded through the fusion of 

other affinity reagents (e.g. DARPins, nanobodies) to SNAP- or Halo-tag via modular cloning 

strategies. The use of defined, recombinant affinity reagents at the core of the PHD technology 

satisfies an urgent need to reduce the use of animal-derived, polyclonal reagents (as highlighted 

e.g. in recent EU directives74). Including secondary covalent capture modules (e.g. 

SpyCatcher/SpyTag, SnpCatcher/SnpTag) adds an extra layer of stable engineerability to the 

system and enables a second dimension of orthogonality for the creation of multifunctional 

hydrogels, while the use of valency-engineering modules allows monomeric proteins to be 



readily assembled into multivalent constructs. Complex multivalent and/or multi-protein 

decorations are accessible from (separate or mixed) solutions of monomers – these decorations 

are assembled on-bead and render cloning of additional multivalent constructs unnecessary. 

Multivalency75,76 and induced proximity77 is a natural mechanism of enhancing and 

manipulating interactions in biological systems by cooperativity,78 most prominently in natural 

antibody biology and the biotherapeutics inspired by it.82  There are no general rules for the 

design of multivalent constructs that take advantage of entropic, avidity or co-localisation 

effects, so the orientation of monomers has to be empirically explored and an experimental 

format to empirically assess the contribution of multivalency is necessary. This fact is 

highlighted in our work by the most potent inducer of apoptosis being a pentavalent antibody 

construct, despite knowledge that the target (TRAIL-R1) is agonised by a trimeric ligand in 

vivo. Typically, multivalent constructs are cloned and expressed as in-frame fusion proteins, 

requiring extensive and often practically difficult cloning (e.g. for sequence-homologous 

repeats that create PCR problems), alongside often expensive and complex mammalian cell 

expression (e.g. in the case of IgG), limiting both the accessibility of protein engineering and 

its throughput. However, with PHD beads judicious choice of valency engineering modules 

can bring about such constructs in multiple permutations simply by incubation instead of 

cloning, once the monomeric modules are available.  

 

Versatility is further boosted by the possibility of photorelease. Steric hindrance and proximity 

to an ill-defined or hydrophobic surface can limit the applicability of protein assays on beads 

(in particular for cell-protein interactions), even though the 3D distribution in PHD beads and 

the solution-like nature of the hydrogel minimise these effects. However, the feature of 

controlled release of the bead-displayed proteins by optical control removes this common 

objection against the use of immobilised proteins in assays (as seen by the release of small 

molecule compounds in OBOC assays79). We show that trivalent scFv has to be released from 

beads in order to potently induce apoptosis. This observation is consistent with the specific 

exclusion of cells from the internal volume of the polyacrylamide hydrogel, an effect also 

observed with yeast cells by Abate et al.70, whilst still allowing large, biologically relevant 

macromolecules (e.g. IgG) to permeate fully. This ‘permeability and exclusion feature’ could 

be taken advantage of, and engineered further, in future applications involving therapeutic 

protein delivery in vivo. For instance, protease sites could be added to modules, enabling the 

tissue-specific release of sequestered/inactive protein drugs80,81. Other future applications to 



take advantage of optical release could include e.g. functional tests with proteins that need to 

be internalised to target intracellular processes or the control of growth factor presentation for 

tissue engineering.  

 

Taken together, the versatility of PHD beads allows an unprecedented degree of freedom in the 

design of bioassay experiments. Straightforward bead-mediated harvesting of proteins from 

lysates, valency control (both at the hydrogel decoration stage and for protein constructs), 

orthogonality of the coupling chemistry (through various tags) and controlled release constitute 

a technology suite capable of simplifying the planning and execution of discovery campaigns 

based on modularity (Figure 7). We have demonstrated the simple reformatting of beads and 

proteins for investigating protein:protein interactions, enzymatic catalysis, bacteriolysis and 

phenotypic assays, but an even wider range of assays and applications is conceivable and take 

advantage of salient features of PHD beads: biocatalysis, in vivo drug delivery, controlled 

release, and sensors. 

 
Figure 7. Overview of a modular “build-an-assay” strategy based on PHD beads. Starting from 
functionalised microbeads (1, see below), choices that define the assay format include the desired 
valency of each single bead as well as the loading of orthogonal protein capture into the system 
(controlled by the input concentrations of BG and CA). Next, one can choose how to capture a desired 
protein (2): either directly as a SNAP- or Halo-tag fusion protein, or via secondary capture modules. 
Secondary capture modules add the capability to specifically capture native or tagged proteins non-
covalently, or to specifically and covalently capture proteins bearing tags, e.g. using the SpyTag-
SpyCatcher or SnpTag-SnpCatcher technologies. At this stage one can also choose to create multivalent 
constructs from monomeric input proteins of interest through the use of valency engineering modules. 
Finally (3), the captured proteins can be tested in on-bead assays (e.g. for their affinity), or released 
from bead in response to irradiation of light, so that the new molecular assemblies can be assayed in 
solution (e.g. for phenotypic cellular assays). Monodisperse beads can be created in microfluidic 

Bead
CA

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Protein capture Application

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devices via water-in-oil emulsions. The design of the microfluidic device and its operation determines 
the bead size. Alternatively, polydisperse emulsions protocols can be used to make beads at the price 
of a broader size distribution. As an alternative to the bead format, functionalised hydrogels can also be 
created on a surface (e.g. for cell culture). 
 

 

EXPERIMENTAL SECTION 

 

Protocol for hydrogel bead synthesis and functionalisation  

(1) The small molecule anchors (methacrylate-PEG-benzylguanine/-chloroalkane; Table S2.1) 

for hydrogel functionalisation were synthesised by mixing one volume of 40 mM BG-PEG-

NH2 (NEB S9150S) or 40 mM chloroalkane-PEG-NH2 (Promega P6741) with one volume of 

40 mM methacrylate-NHS (Sigma 730300) overnight at room temperature at 400 rpm in the 

presence of a 1.5-fold molar excess of triethylamine (Sigma 471283). All solutions were 

prepared fresh from powder in anhydrous DMSO (Merck 276855) except triethylamine which 

was added from neat stock. After overnight incubation the reaction was quenched with 3 

volumes of 100 mM Tris-HCl (pH 8.0) and rolled 1 hour at room temperature, yielding a final 

concentration of 5 mM product.  

(2) To prepare functionalised beads, unpolymerised hydrogel mix (10 mM Tris-HCl (pH 7.6), 

1 mM EDTA, 15 mM NaCl, 6.2% (v/v) acrylamide, 0.18% (v/v) bisacrylamide, 0.3% (w/v) 

ammonium persulfate) containing the small molecule anchors was encapsulated in oil (008- 

Fluorosurfactant 1.35% w/w, RAN Biotechnologies, TEMED 0.4 % v/v in HFE-7500 (3M 

Novec)) in a microfluidic droplet generator (Figure S2.1), as previously described27. After 

encapsulation the emulsion was incubated overnight at 65 °C under mineral oil. The next day, 

polymerised hydrogel beads were recovered by breaking the emulsion with 800 µL wash buffer 

(100 mM Tris-HCl, 0.1 % Tween-20), and 200 µL 1H,1H,2H,2H-perfluorooctanol (PFO, 97%, 

Alfa Aesar). The tube was inverted several times and briefly centrifuged for 5 seconds at 100 

g before recovering the aqueous bead-containing phase into a fresh tube. Large polyacrylamide 

particles were removed by passing the mixture through a 10 µm filter (CellTrics) for 30 seconds 

at 200 g before using a haemocytometer (KOVA Glasstic) to determine the ‘concentration’ of 

beads in the suspension. These beads are stable at 4 °C for many months. For all assays beads 

are typically incubated and washed in buffer (100 mM Tris-HCl, 0.1 % Tween-20). In other 

buffers and in unbuffered water, the bead pellet after centrifugation can be difficult to identify. 



(3) SNAP-tag/Halo-tag fusion proteins were captured by incubating with a defined number of 

beads for >30 minutes with rolling in wash buffer. After protein capture, beads were typically 

washed three times in wash buffer. Subsequent capture of tagged or untagged proteins was 

performed in the same manner.  

(4) On-bead photocleavage was carried out by attaching PCR tubes containing beads to a 

cooled metal block and exposing to 405 nm light at full power from a LED (M405L2 Thorlabs) 

driven by LEDD1b (Thorlabs).  

 

ASSOCIATED CONTENT   

The Supporting Information is available free of charge at https://pubs.acs.org/doi/ and contains 

experimental results (HPLC analysis of BG formation, functionalisation of polydisperse PHD 

beads, valency calculations, cell lysate saturation binding and modular Gibson assembly 

design) and experimental procedures (benzylguanine analysis via HPLC), microfluidics and 

bead handling, protein expression and purification, molecular biology of module construction, 

cell culture, biological assays)  

 

AUTHOR INFORMATION 

CORRESPONDING AUTHOR 

* Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, 

CB2 1GA, UK; email: fh111@cam.ac.uk. 

Present Addresses 
† Alchemab Therapeutics Ltd., 55-56 Russel Square, London, WC1B 4HP, UK. 

 

AUTHOR CONTRIBUTIONS  

T.F., J.D.R. and F.H. designed research, T.F., J.D.R., C.M. and T.N.K. performed research, 

T.F., J.D.R. and F.H. analysed data, R.M. and F.H. directed research and T.F., J.D.R. and F.H. 

wrote the manuscript with feedback from all authors. All authors have given approval to the 

final version of the manuscript. 

 

FUNDING SOURCES 



BBSRC iCASE studentship (to T. F., BB/M016692/1) and BBSRC-CTP studentship (to J.D.R, 

BB/R505055/1). FH is an ERC Advanced Investigator (695669).  

 

ACKNOWLEDGMENT  

The authors thank members of the Hollfelder group for comments on the manuscript, and Dr. 

J.D.F. Schnettler-Fernandez for the P91 vector and substrate. Thanks also to Dr. Joana Cerveira 

of the Cambridge BioPath Flow cytometry facility, Camilla Trevor (AstraZeneca), Josie 

Holstein (University of Cambridge) and the Core Tissue Culture Facility (AstraZeneca) for 

their guidance and practical support. 

 

References 

(1)  Li, J.; Mooney, D. J. Designing Hydrogels for Controlled Drug Delivery. Nat. Rev. 
Mater. 2016, 1 (12), 16071. https://doi.org/10.1038/natrevmats.2016.71. 

(2)  Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and 
How. Chem Soc Rev 2013, 42 (15), 6223–6235. https://doi.org/10.1039/C3CS60075K. 

(3)  Thakur, M.; Medintz, I. L.; Walper, S. A. Enzymatic Bioremediation of 
Organophosphate Compounds—Progress and Remaining Challenges. Front. Bioeng. 
Biotechnol. 2019, 7, 289. https://doi.org/10.3389/fbioe.2019.00289. 

(4)  Silva, A. K. A.; Richard, C.; Bessodes, M.; Scherman, D.; Merten, O.-W. Growth Factor 
Delivery Approaches in Hydrogels. Biomacromolecules 2009, 10 (1), 9–18. 
https://doi.org/10.1021/bm801103c. 

(5)  McConnell, S. J.; Dinh, T.; Le, M.-H.; Spinella, D. G. Biopanning Phage Display 
Libraries Using Magnetic Beads Vs. Polystyrene Plates. BioTechniques 1999, 26 (2), 
208–214. https://doi.org/10.2144/99262bm06. 

(6)  Aydin, S. A Short History, Principles, and Types of ELISA, and Our Laboratory 
Experience with Peptide/Protein Analyses Using ELISA. Peptides 2015, 72, 4–15. 
https://doi.org/10.1016/j.peptides.2015.04.012. 

(7)  Jason-Moller, L.; Murphy, M.; Bruno, J. Overview of Biacore Systems and Their 
Applications. Curr. Protoc. Protein Sci. 2006, 45 (1). 
https://doi.org/10.1002/0471140864.ps1913s45. 

(8)  De Santis, P.; Meyer, L.-E.; Kara, S. The Rise of Continuous Flow Biocatalysis – 
Fundamentals, Very Recent Developments and Future Perspectives. React. Chem. Eng. 
2020, 5 (12), 2155–2184. https://doi.org/10.1039/D0RE00335B. 

(9)  Schmieg, B.; Döbber, J.; Kirschhöfer, F.; Pohl, M.; Franzreb, M. Advantages of 
Hydrogel-Based 3D-Printed Enzyme Reactors and Their Limitations for Biocatalysis. 
Front. Bioeng. Biotechnol. 2019, 6, 211. https://doi.org/10.3389/fbioe.2018.00211. 

(10)  Peschke, T.; Bitterwolf, P.; Gallus, S.; Hu, Y.; Oelschlaeger, C.; Willenbacher, N.; 
Rabe, K. S.; Niemeyer, C. M. Self-Assembling All-Enzyme Hydrogels for Flow 
Biocatalysis. Angew. Chem. Int. Ed. 2018, 57 (52), 17028–17032. 
https://doi.org/10.1002/anie.201810331. 

(11)  Patra, J. K.; Das, G.; Fraceto, L. F.; Campos, E. V. R.; Rodriguez-Torres, M. del P.; 
Acosta-Torres, L. S.; Diaz-Torres, L. A.; Grillo, R.; Swamy, M. K.; Sharma, S.;      et al. 



Nano Based Drug Delivery Systems: Recent Developments and Future Prospects. J. 
Nanobiotechnology 2018, 16 (1), 71. https://doi.org/10.1186/s12951-018-0392-8. 

(12)  Laustsen, A. H.; Lauridsen, L. P.; Lomonte, B.; Andersen, M. R.; Lohse, B. Pitfalls to 
Avoid When Using Phage Display for Snake Toxins. Toxicon 2017, 126, 79–89. 
https://doi.org/10.1016/j.toxicon.2016.12.010. 

(13)  Rodrigues, R. C.; Ortiz, C.; Berenguer-Murcia, Á.; Torres, R.; Fernández-Lafuente, R. 
Modifying Enzyme Activity and Selectivity by Immobilization. Chem Soc Rev 2013, 42 
(15), 6290–6307. https://doi.org/10.1039/C2CS35231A. 

(14)  Hoarau, M.; Badieyan, S.; Marsh, E. N. G. Immobilized Enzymes: Understanding 
Enzyme – Surface Interactions at the Molecular Level. Org. Biomol. Chem. 2017, 15 
(45), 9539–9551. https://doi.org/10.1039/C7OB01880K. 

(15)  Chaparro Sosa, A. F.; Bednar, R. M.; Mehl, R. A.; Schwartz, D. K.; Kaar, J. L. Faster 
Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity. J. 
Am. Chem. Soc. 2021, jacs.1c02375. https://doi.org/10.1021/jacs.1c02375. 

(16)  Pezzi, H. M.; Niles, D. J.; Schehr, J. L.; Beebe, D. J.; Lang, J. M. Integration of 
Magnetic Bead-Based Cell Selection into Complex Isolations. ACS Omega 2018, 3 (4), 
3908–3917. https://doi.org/10.1021/acsomega.7b01427. 

(17)  Tan, X.; Letendre, J. H.; Collins, J. J.; Wong, W. W. Synthetic Biology in the Clinic: 
Engineering Vaccines, Diagnostics, and Therapeutics. Cell 2021, 184 (4), 881–898. 
https://doi.org/10.1016/j.cell.2021.01.017. 

(18)  Fantoni, N. Z.; El-Sagheer, A. H.; Brown, T. A Hitchhiker’s Guide to Click-Chemistry 
with Nucleic Acids. Chem. Rev. 2021, acs.chemrev.0c00928. 
https://doi.org/10.1021/acs.chemrev.0c00928. 

(19)  Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function 
from a Few Good Reactions. Angew Chem Int Ed 2001, 18. 

(20)  Modica, J. A.; Iderzorig, T.; Mrksich, M. Design and Synthesis of Megamolecule 
Mimics of a Therapeutic Antibody. J. Am. Chem. Soc. 2020, jacs.0c05093. 
https://doi.org/10.1021/jacs.0c05093. 

(21)  Divine, R.; Dang, H. V.; Ueda, G.; Fallas, J. A.; Vulovic, I.; Sheffler, W.; Saini, S.; 
Zhao, Y. T.; Raj, I. X.; Morawski, P. A. et al     . Designed Proteins Assemble 
Antibodies into Modular Nanocages. Science 2021, 372 (6537), eabd9994. 
https://doi.org/10.1126/science.abd9994. 

(22)  Anuar, I. N. A. K.; Banerjee, A.; Keeble, A. H.; Carella, A.; Nikov, G. I.; Howarth, M. 
Spy&Go Purification of SpyTag-Proteins Using Pseudo-SpyCatcher to Access an 
Oligomerization Toolbox. Nat. Commun. 2019, 10 (1), 1–13. 
https://doi.org/10.1038/s41467-019-09678-w. 

(23)  Zhang, W.; Lohman, A. W.; Zhuravlova, Y.; Lu, X.; Wiens, M. D.; Hoi, H.; Yaganoglu, 
S.; Mohr, M. A.; Kitova, E. N.; Klassen, J. S.           et al.  Optogenetic Control with a 
Photocleavable Protein, PhoCl. Nat. Methods 2017, 14 (4), 391–394. 
https://doi.org/10.1038/nmeth.4222. 

(24)  Shadish, J. A.; Strange, A. C.; DeForest, C. A. Genetically Encoded Photocleavable 
Linkers for Patterned Protein Release from Biomaterials. J. Am. Chem. Soc. 2019, 141 
(39), 15619–15625. https://doi.org/10.1021/jacs.9b07239. 

(25)  Götzke, H.; Kilisch, M.; Martínez-Carranza, M.; Sograte-Idrissi, S.; Rajavel, A.; 
Schlichthaerle, T.; Engels, N.; Jungmann, R.; Stenmark, P.; Opazo, F.           et al. The 
ALFA-Tag Is a Highly Versatile Tool for Nanobody-Based Bioscience Applications. 
Nat. Commun. 2019, 10 (1), 4403. https://doi.org/10.1038/s41467-019-12301-7. 

(26)  Mondal, S.; Das, S.; Nandi, A. K. A Review on Recent Advances in Polymer and 
Peptide Hydrogels. Soft Matter 2020, 16 (6), 1404–1454. 
https://doi.org/10.1039/C9SM02127B. 



(27)  Klein, A. M.; Mazutis, L.; Akartuna, I.; Tallapragada, N.; Veres, A.; Li, V.; Peshkin, L.; 
Weitz, D. A.; Kirschner, M. W. Droplet Barcoding for Single-Cell Transcriptomics 
Applied to Embryonic Stem Cells. Cell 2015, 161 (5), 1187–1201. 
https://doi.org/10.1016/j.cell.2015.04.044. 

(28)  Tibbitt, M. W.; Anseth, K. S. Hydrogels as Extracellular Matrix Mimics for 3D Cell 
Culture. 2010, 16. 

(29)  Lai, S. N.; Zhou, X.; Ouyang, X.; Zhou, H.; Liang, Y.; Xia, J.; Zheng, B. Artificial Cells 
Capable of Long-Lived Protein Synthesis by Using Aptamer Grafted Polymer Hydrogel. 
ACS Synth. Biol. 2020, acssynbio.9b00338. https://doi.org/10.1021/acssynbio.9b00338. 

(30)  Zayats, M.; Brenner, A. J.; Searson, P. C. Protein Imprinting in Polyacrylamide-Based 
Gels. Biomaterials 2014, 35 (30), 8659–8668. 
https://doi.org/10.1016/j.biomaterials.2014.05.079. 

(31)  Hammer, J. A.; West, J. L. Chemically Orthogonal Protein Ligation Domains for 
Independent Control of Hydrogel Modification with Adhesive Ligands and Growth 
Factors. Bioconjug. Chem. 2020, acs.bioconjchem.0c00405. 
https://doi.org/10.1021/acs.bioconjchem.0c00405. 

(32)  Lim, S.; Jung, G. A.; Muckom, R. J.; Glover, D. J.; Clark, D. S. Engineering 
Bioorthogonal Protein–Polymer Hybrid Hydrogel as a Functional Protein 
Immobilization Platform. Chem. Commun. 2019, 55 (6), 806–809. 
https://doi.org/10.1039/C8CC08720B. 

(33)  Denisin, A. K.; Pruitt, B. L. Tuning the Range of Polyacrylamide Gel Stiffness for 
Mechanobiology Applications. ACS Appl. Mater. Interfaces 2016, 8 (34), 21893–21902. 
https://doi.org/10.1021/acsami.5b09344. 

(34)  Kandow, C. E.; Georges, P. C.; Janmey, P. A.; Beningo, K. A. Polyacrylamide 
Hydrogels for Cell Mechanics: Steps Toward Optimization and Alternative Uses. In 
Methods in Cell Biology; Cell Mechanics; Academic Press, 2007; Vol. 83, pp 29–46. 
https://doi.org/10.1016/S0091-679X(07)83002-0. 

(35)  (a) Wenger, Y.; Schneider, R. J.; Reddy, G. R.; Kopelman, R.; Jolliet, O.; Philbert, M. 
A. Tissue Distribution and Pharmacokinetics of Stable Polyacrylamide Nanoparticles 
Following Intravenous Injection in the Rat. Toxicol. Appl. Pharmacol. 2011, 251 (3), 
181–190. https://doi.org/10.1016/j.taap.2010.11.017.  (b) Shui, L.; van den Berg, A.; 
Eijkel, J. C. T. Scalable attoliter monodisperse droplet formation using multiphase nano-
microfluidics Microfluidic and Nanofluidics 2011, 11, 87-92. 
https://doi.org/10.1007/s10404-011-0776-7 

(36)  Kuruppuarachchi, M.; Savoie, H.; Lowry, A.; Alonso, C.; Boyle, R. W. Polyacrylamide 
Nanoparticles as a Delivery System in Photodynamic Therapy. Mol. Pharm. 2011, 8 (3), 
920–931. https://doi.org/10.1021/mp200023y. 

(37)  Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. A 
General Method for the Covalent Labeling of Fusion Proteins with Small Molecules in 
Vivo. Nat. Biotechnol. 2003, 21 (1), 86–89. https://doi.org/10.1038/nbt765. 

(38)  Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, 
C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M. et al. HaloTag: A Novel Protein 
Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3 
(6), 373–382. https://doi.org/10.1021/cb800025k. 

(39)  Kindermann, M.; George, N.; Johnsson, N.; Johnsson, K. Covalent and Selective 
Immobilization of Fusion Proteins. J. Am. Chem. Soc. 2003, 125 (26), 7810–7811. 
https://doi.org/10.1021/ja034145s. 

(40)  Döbber, J.; Pohl, M.; Ley, S. V.; Musio, B. Rapid, Selective and Stable HaloTag- Lb 
ADH Immobilization Directly from Crude Cell Extract for the Continuous Biocatalytic 



Production of Chiral Alcohols and Epoxides. React. Chem. Eng. 2018, 3 (1), 8–12. 
https://doi.org/10.1039/C7RE00173H. 

(41)  Abate, A. R.; Chen, C.-H.; Agresti, J. J.; Weitz, D. A. Beating Poisson Encapsulation 
Statistics Using Close-Packed Ordering. Lab. Chip 2009, 9 (18), 2628. 
https://doi.org/10.1039/b909386a. 

(42)  Diamante, L.; Gatti-Lafranconi, P.; Schaerli, Y.; Hollfelder, F. In Vitro Affinity 
Screening of Protein and Peptide Binders by Megavalent Bead Surface Display. Protein 
Eng. Des. Sel. 2013, 26 (10), 713–724. https://doi.org/10.1093/protein/gzt039. 

(43)  Steiner, D.; Forrer, P.; Plückthun, A. Efficient Selection of DARPins with Sub-
Nanomolar Affinities Using SRP Phage Display. J. Mol. Biol. 2008, 382 (5), 1211–
1227. https://doi.org/10.1016/j.jmb.2008.07.085. 

(44)  Gilbreth, R. N.; Truong, K.; Madu, I.; Koide, A.; Wojcik, J. B.; Li, N.-S.; Piccirilli, J. 
A.; Chen, Y.; Koide, S. Isoform-Specific Monobody Inhibitors of Small Ubiquitin-
Related Modifiers Engineered Using Structure-Guided Library Design. Proc. Natl. 
Acad. Sci. 2011, 108 (19), 7751–7756. https://doi.org/10.1073/pnas.1102294108. 

(45)  Keeble, A. H.; Howarth, M. Power to the Protein: Enhancing and Combining Activities 
Using the Spy Toolbox. Chem. Sci. 2020, 11 (28), 7281–7291. 
https://doi.org/10.1039/D0SC01878C. 

(46)  Tan, T. K.; Rijal, P.; Rahikainen, R.; Keeble, A. H.; Schimanski, L.; Hussain, S.; 
Harvey, R.; Hayes, J. W. P.; Edwards, J     . C.; McLean, R. K. et al.. A COVID-19 
Vaccine Candidate Using SpyCatcher Multimerization of the SARS-CoV-2 Spike 
Protein Receptor-Binding Domain Induces Potent Neutralising Antibody Responses; 
Nat Commun 12, 542 (2021). https://doi.org/10.1038/s41467-020-20654-7 

(47)  Schoene, C.; Fierer, J. O.; Bennett, S. P.; Howarth, M. SpyTag/SpyCatcher Cyclization 
Confers Resilience to Boiling on a Mesophilic Enzyme. Angew. Chem. Int. Ed. 2014, 53 
(24), 6101–6104. https://doi.org/10.1002/anie.201402519. 

(48)  Veggiani, G.; Nakamura, T.; Brenner, M. D.; Gayet, R. V.; Yan, J.; Robinson, C. V.; 
Howarth, M. Programmable Polyproteams Built Using Twin Peptide Superglues. Proc. 
Natl. Acad. Sci. 2016, 113 (5), 1202–1207. https://doi.org/10.1073/pnas.1519214113. 

(49)  Liu, Z.; Zhou, H.; Wang, W.; Tan, W.; Fu, Y.-X.; Zhu, M. A Novel Method for 
Synthetic Vaccine Construction Based on Protein Assembly. Sci. Rep. 2015, 4 (1), 
7266. https://doi.org/10.1038/srep07266. 

(50)  Keeble, A. H.; Banerjee, A.; Ferla, M. P.; Reddington, S. C.; Anuar, I. N. A. K.; 
Howarth, M. Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze 
Membrane Dynamics. Angew. Chem. Int. Ed. 2017, 56 (52), 16521–16525. 
https://doi.org/10.1002/anie.201707623. 

(51)  Gao, X.; Lyu, S.; Li, H. Decorating a Blank Slate Protein Hydrogel: A General and 
Robust Approach for Functionalizing Protein Hydrogels. Biomacromolecules 2017, 18 
(11), 3726–3732. https://doi.org/10.1021/acs.biomac.7b01369. 

(52)  Gai, S. A.; Wittrup, K. D. Yeast Surface Display for Protein Engineering and 
Characterization. Curr. Opin. Struct. Biol. 2007, 17 (4), 467–473. 
https://doi.org/10.1016/j.sbi.2007.08.012. 

(53)  Akerström, B.; Björck, L. A Physicochemical Study of Protein G, a Molecule with 
Unique Immunoglobulin G-Binding Properties. J. Biol. Chem. 1986, 261 (22), 10240–
10247. https://doi.org/10.1016/S0021-9258(18)67515-5. 

(54)  Huovinen, T.; Lindenburg, L.; Minter, R.; Hollfelder, F. Multiplexed Affinity 
Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent 
Capture on Suspension Bead Arrays. Anal. Chem. 2021, 93 (4), 2166–2173. 
https://doi.org/10.1021/acs.analchem.0c03992. 



(55)  Colin, P.-Y.; Kintses, B.; Gielen, F.; Miton, C. M.; Fischer, G.; Mohamed, M. F.; 
Hyvönen, M.; Morgavi, D. P.; Janssen, D. B.; Hollfelder, F. Ultrahigh-Throughput 
Discovery of Promiscuous Enzymes by Picodroplet Functional Metagenomics. Nat. 
Commun. 2015, 6 (1). https://doi.org/10.1038/ncomms10008. 

(56)  Silverman, A. D.; Karim, A. S.; Jewett, M. C. Cell-Free Gene Expression: An Expanded 
Repertoire of Applications. Nat. Rev. Genet. 2020, 21 (3), 151–170. 
https://doi.org/10.1038/s41576-019-0186-3. 

(57)  Holstein, J. M.; Gylstorff, C.; Hollfelder, F. Cell-Free Directed Evolution of a Protease 
in Microdroplets at Ultrahigh Throughput. ACS Synth. Biol. 2021, 10 (2), 252–257. 
https://doi.org/10.1021/acssynbio.0c00538. 

(58)  Lee, A. J.; Wang, S.; Meredith, H. R.; Zhuang, B.; Dai, Z.; You, L. Robust, Linear 
Correlations between Growth Rates and β-Lactam–Mediated Lysis Rates. Proc. Natl. 
Acad. Sci. 2018, 115 (16), 4069–4074. https://doi.org/10.1073/pnas.1719504115. 

(59)  Butler, K.; English, A. R.; Ray, V. A.; Timreck, A. E. Carbenicillin: Chemistry and 
Mode of Action. J. Infect. Dis. 1970, 122, S1–S8. 

(60)  Qin, M. Hydrogels With Tunable Mechanical Properties Based on Photocleavable 
Proteins. Front. Chem. 2020, 8, 9. 

(61) Lu, X.; Campbell, R. E. Improved Photocleavable Proteins with Faster and More 
Efficient Dissociation. Chem. Sci., 2021,12, 9658-9672 
https://doi.org/10.1039/D1SC01059J 

(62)  LeBlanc, H. N.; Ashkenazi, A. Apo2L/TRAIL and Its Death and Decoy Receptors. Cell 
Death Differ. 2003, 10 (1), 66–75. https://doi.org/10.1038/sj.cdd.4401187. 

(63)  Huang, Y.; Bharill, S.; Karandur, D.; Peterson, S. M.; Marita, M.; Shi, X.; Kaliszewski, 
M. J.; Smith, A. W.; Isacoff, E. Y.; Kuriyan, J. Molecular Basis for Multimerization in 
the Activation of the Epidermal Growth Factor Receptor. eLife 2016, 5, e14107. 
https://doi.org/10.7554/eLife.14107. 

(64)  Dobson, C. L.; Main, S.; Newton, P.; Chodorge, M.; Cadwallader, K.; Humphreys, R. 
C.; Albert, V.; Vaughan, T. J.; Minter, R. R.; Edwards, B. M. Human Monomeric 
Antibody Fragments to TRAIL-R1 and TRAIL-R2 That Display Potent in Vitro 
Agonism. mAbs 2009, 1 (6), 552–562. https://doi.org/10.4161/mabs.1.6.10057. 

(65)  Pukac, L.; Kanakaraj, P.; Humphreys, R.; Alderson, R.; Bloom, M.; Sung, C.; 
Riccobene, T.; Johnson, R.; Fiscella, M.; Mahoney, A. et al. HGS-ETR1, a Fully 
Human TRAIL-Receptor 1 Monoclonal Antibody, Induces Cell Death in Multiple 
Tumour Types in Vitro and in Vivo. Br. J. Cancer 2005, 92 (8), 1430–1441. 
https://doi.org/10.1038/sj.bjc.6602487. 

(66)  von Pawel, J.; Harvey, J. H.; Spigel, D. R.; Dediu, M.; Reck, M.; Cebotaru, C. L.; 
Humphreys, R. C.; Gribbin, M. J.; Fox, N. L.; Camidge, D. R. Phase II Trial of 
Mapatumumab, a Fully Human Agonist Monoclonal Antibody to Tumor Necrosis 
Factor-Related Apoptosis-Inducing Ligand Receptor 1 (TRAIL-R1), in Combination 
With Paclitaxel and Carboplatin in Patients With Advanced Non–Small-Cell Lung 
Cancer. Clin. Lung Cancer 2014, 15 (3), 188-196.e2. 
https://doi.org/10.1016/j.cllc.2013.12.005. 

(67)  Trarbach, T.; Moehler, M.; Heinemann, V.; Köhne, C.-H.; Przyborek, M.; Schulz, C.; 
Sneller, V.; Gallant, G.; Kanzler, S. Phase II Trial of Mapatumumab, a Fully Human 
Agonistic Monoclonal Antibody That Targets and Activates the Tumour Necrosis 
Factor Apoptosis-Inducing Ligand Receptor-1 (TRAIL-R1), in Patients with Refractory 
Colorectal Cancer. Br. J. Cancer 2010, 102 (3), 506–512. 
https://doi.org/10.1038/sj.bjc.6605507. 



(68)  de Miguel, D.; Lemke, J.; Anel, A.; Walczak, H.; Martinez-Lostao, L. Onto Better 
TRAILs for Cancer Treatment. Cell Death Differ. 2016, 23 (5), 733–747. 
https://doi.org/10.1038/cdd.2015.174. 

(69)  Huet, H. A.; Growney, J. D.; Johnson, J. A.; Li, J.; Bilic, S.; Ostrom, L.; Zafari, M.; 
Kowal, C.; Yang, G.; Royo, A. et al     . Multivalent Nanobodies Targeting Death 
Receptor 5 Elicit Superior Tumor Cell Killing through Efficient Caspase Induction. 
mAbs 2014, 6 (6), 1560–1570. https://doi.org/10.4161/19420862.2014.975099. 

(70)  Hatori, M. N.; Kim, S. C.; Abate, A. R. Particle-Templated Emulsification for 
Microfluidics-Free Digital Biology. Anal. Chem. 2018, 90 (16), 9813–9820. 
https://doi.org/10.1021/acs.analchem.8b01759. 

(71)  Marconi, M.; Ascione, B.; Ciarlo, L.; Vona, R.; Garofalo, T.; Sorice, M.; Gianni, A. M.; 
Locatelli, S. L.; Carlo-Stella, C.; Malorni, W      et al. Constitutive Localization of DR4 
in Lipid Rafts Is Mandatory for TRAIL-Induced Apoptosis in B-Cell Hematologic 
Malignancies. Cell Death Dis. 2013, 4 (10), e863–e863. 
https://doi.org/10.1038/cddis.2013.389. 

(72)  Gautier, A.; Juillerat, A.; Heinis, C.; Corrêa, I. R.; Kindermann, M.; Beaufils, F.; 
Johnsson, K. An Engineered Protein Tag for Multiprotein Labeling in Living Cells. 
Chem. Biol. 2008, 15 (2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007. 

(73)  Kimmel, B. R.; Mrksich, M. Development of an Enzyme-Inhibitor Reaction Using 
Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly. Chem. 
– Eur. J. 2021, 27 (71), 17843–17848. https://doi.org/10.1002/chem.202103059. 

(74)  Gray, A. C.; Bradbury, A. R. M.; Knappik, A.; Plückthun, A.; Borrebaeck, C. A. K.; 
Dübel, S. Animal-Derived-Antibody Generation Faces Strict Reform in Accordance 
with European Union Policy on Animal Use. Nat. Methods 2020, 17 (8), 755–756. 
https://doi.org/10.1038/s41592-020-0906-9. 

(75)  Fasting, C.; Schalley, C. A.; Weber, M.; Seitz, O.; Hecht, S.; Koksch, B.; Dernedde, J.; 
Graf, C.; Knapp, E.-W.; Haag, R. Multivalency as a Chemical Organization and Action 
Principle. Angew. Chem. Int. Ed. 2012, 51 (42), 10472–10498. 
https://doi.org/10.1002/anie.201201114. 

(76)  Varner, C. T.; Rosen, T.; Martin, J. T.; Kane, R. S. Recent Advances in Engineering 
Polyvalent Biological Interactions. Biomacromolecules 2015, 16 (1), 43–55. 
https://doi.org/10.1021/bm5014469. 

(77)  Stanton, B. Z.; Chory, E. J.; Crabtree, G. R. Chemically Induced Proximity in Biology 
and Medicine. Science 2018, 359 (6380), eaao5902. 
https://doi.org/10.1126/science.aao5902. 

(78)  Hunter, C. A.; Anderson, H. L. What Is Cooperativity? Angew. Chem. Int. Ed. 2009, 48 
(41), 7488–7499. https://doi.org/10.1002/anie.200902490. 

(79)  Liu, T.; Qian, Z.; Xiao, Q.; Pei, D. High-Throughput Screening of One-Bead-One-
Compound Libraries: Identification of Cyclic Peptidyl Inhibitors against 
Calcineurin/NFAT Interaction. ACS Comb. Sci. 2011, 13 (5), 537–546. 
https://doi.org/10.1021/co200101w. 

(80)  Autio, K. A.; Boni, V.; Humphrey, R. W.; Naing, A. Probody Therapeutics: An 
Emerging Class of Therapies Designed to Enhance On-Target Effects with Reduced 
Off-Tumor Toxicity for Use in Immuno-Oncology. Clin. Cancer Res. 2020, 26 (5), 
984–989. https://doi.org/10.1158/1078-0432.CCR-19-1457. 

(81)  Secret, E.; Crannell, K. E.; Kelly, S. J.; Villancio-Wolter, M.; Andrew, J. S. Matrix 
Metalloproteinase-Sensitive Hydrogel Microparticles for Pulmonary Drug Delivery of 
Small Molecule Drugs or Proteins. J. Mater. Chem. B 2015, 3 (27), 5629–5634. 
https://doi.org/10.1039/C5TB00443H. 



(82)  Oostindie, S. C.; Lazar, G. A.; Schuurman, J.; Parren, P. W. H. I.  Avidity in antibody 
effector functions and biotherapeutic drug design. Nature Reviews Drug Discovery 
2022, in press. https://doi.org/10.1038/s41573-022-00501-8.  

  



Table of Contents Graphics  

 

 
      

 

Synopsis 

 

‘Plug and play’ display of up to ~109 protein molecules attached to monodisperse beads affords 

control over valency and release, creating a platform for analysis and use of proteins. 

Bead
CA

BG
BG BG

CA
CA

hv1-6

1-6

Halo
POI

SN
AP

PO
I

Fluorescent 
m

odule

Non-covalent capture

modules

Covalent capture

modules

Photocontrolled multivalent 
engineering modules

SN
AP

-P
ro

te
in

 G
:

IgG

SNAP-I19:

Human IgG

SNAP-YMB:

SUMO-tag

SNAP-SpyTag:SpyCatcher

SNAP-SnpTag:

SnpCatcher

SNAP-SpyC:

SpyT

SNAP-SnpC:

SnpT
Halo-SnpC:

SnpT

SN
AP-PhoCl-S

pyC 1-6
:

Sp
yT

SN
AP

-G
FP

M
odule design