Mechanoresponsive and recyclable biocatalytic sponges from enzyme-polymer surfactant conjugates and nanoparticles

Article abstract of DOI:10.1039/c8ra08221a

The development of multifunctional hybrid biomaterials is an important area of focus in tissue engineering, drug delivery, biocatalysis, and biosensing applications. Combining bioconjugation methodology with ice templating technique, we show here the deve

Full text of DOI:10.1039/c8ra08221a

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Mechanoresponsive and recyclable biocatalytic
sponges from enzyme-polymer surfactant
conjugates and nanoparticles†
Cite this: RSC Adv., 2018, 8, 39029
*
Mehak Jain, Rutuja G. Vaze, Suraj C. Ugrani and Kamendra P. Sharma
The development of multifunctional hybrid biomaterials is an important area of focus in tissue engineering,
drug delivery, biocatalysis, and biosensing applications. Combining bioconjugation methodology with ice
templating technique, we show here the development of a new class of multifunctional and biocatalytic
scaffold-like spongy material fabricated from an aqueous solution of enzyme-polymer surfactant
(enzyme-PS) core–shell conjugates, and polyethyleneimine (PEI) coated silica/silk nanoparticles. The
generality of this process is demonstrated by the fabrication of biocatalytic sponges comprising PEI
coated nanoparticles and core–shell conjugates of alkaline phosphatase (ALP-PS), or glucose oxidase
(GOx-PS), and horseradish peroxidase (HRP-PS). We show that ALP-PS conjugate driven biocatalytic
transformations can be simply achieved by saturating the highly porous, and manoeuvrable sponges with
the p-nitrophenyl phosphate substrate solution. Subsequently, the compressible and elastic property of
the sponge can be utilized for the extrusion of the product, p-nitrophenol, by applying controlled and
normal mechanical stress. Further, the sponges can be washed and recycled upto ten times, with
approximately 67% retention of initial biocatalytic activity. Interestingly, the ALP-PS conjugate based
sponges exhibit mechanoresponsive catalytic behaviour; the amount of product obtained over 25
minutes of reaction time can be increased by approx. 8 times by compressing-decompressing the
sponge after every 15 seconds. This is attributed to the change in mass transfer and diffusion of the
substrate within the porous channels of the sponge. We also highlight the importance of bioconjugation
of enzymes for fabricating such sponges; our results show that, whilst the native enzymes either
denature or are leached away during the fabrication/biocatalytic usage, their enzyme-PS conjugate
counterparts integrate efficiently to form sturdy, robust, highly catalytic, and recyclable sponge material.
Received 4th October 2018
Accepted 12th November 2018
DOI: 10.1039/c8ra08221a
rsc.li/rsc-advances
biomaterials can be fabricated by combining proteins/enzymes
with auxiliary functionalities like polymers, nanoparticles, and
other molecules.6–8 The auxiliary components provide structural,
1. Introduction
Nature exhibits the supreme ability to design and develop multi-
functional materials.1 This is usually achieved using nucleic acids,
lipids, proteins/enzymes, or carbohydrates as the building blocks,
which can self-assemble to form variety of hierarchical and
ordered structures ranging from cell membranes to tissues. These
materials show adaptive and responsive behaviour, provide struc-
tural and mechanical stability, and also respond by an automated
and controlled feedback towards any chemical, magnetic or an
optical stimuli. With the necessity of addressing health and envi-
ronment related problems, the synthetic design of multifunctional
hybrid materials that nd applications in drug delivery, tissue
engineering, catalysis, bioelectronics, and biosensing has become
the primary goal for many researchers working at the interface of
materials science, chemistry, and biology.2–5 Oen hybrid
mechanical, and other responsive functionalities to the hybrid
biomaterial, while protein imparts specicity and selectivity. In
this context the synergistic unication of these multiple func-
tionalities (proteins/enzymes, nanoparticles, or polymers) is highly
important. The integration of nanoparticles (providing various
properties like higher modulus, photonic, electronic, magnetic
etc.) with proteins can lead to hybrid biomaterials which exhibit
unique synergistic properties, and superior functions.9 Such
hybrid materials have been fabricated in the form of 2D or 3D
ordered assemblies in solutions or have been graed on surfaces
for applications in bioanalytics or bioelectronics devices.7
The most commonly adopted methodology of combining
native proteins with nanoparticles generally involves physical
adsorption or covalent linkage mediated immobilization of the
former on the surface of the later.10–12 However, proteins having
a biological function (enzymes) can undergo denaturation upon
direct immobilization on nanoparticles due to restriction in
conformational freedom, and unfolding caused by covalent or
Department of Chemistry, Indian Institute of Technology Bombay, Powai,
Mumbai-400076, India. E-mail: k.sharma@chem.iitb.ac.in
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra08221a
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non-covalent interactions with the high energy nanoparticle 1,3-propanediamine (purity $ 98%, 39 380, DMAPA), 1-ethyl-3-(3-
surface, hence losing its functionality.13,14 Various methods dimethyl aminopropyl carbodiimide) (purity $ 97%, 39 391,
have been utilized to alleviate this problem, however, a generic EDC), poly(ethylene glycol)4-nonylphenyl 3-sulfopropyl ether
methodology remains to be explored. Moreover, for the potassium salt (473 197, polymer surfactant; PS), LUDOX LS
nanoparticle-enzyme based hybrid biomaterial to be used as an colloidal silica (30% w/w in water, 420 808, silica NPs), Poly-
efficient tissue engineering material, biocatalyst, or a biosensor ethylenimine, branched (average Mw 25 kDa, 408 727, PEI) and
it should overcome the problems related to substrate diffusion. poly(ethylene glycol)diglycidyl ether (475 696, PEGDE) were
Therefore,
a two-step approach involving (i) developing procured from Sigma Aldrich. Peroxidase (from Horseradish,
enzymes with enhanced stability towards denaturation, and (ii) lyophilized powder, $ 139 units per mg, 73 292, HRP) glucose
integrating these with an immobilization matrix which provides oxidase (from Aspergillus niger, lyophilized powder, $ 100 units
features such as porosity (for unrestricted access of the per mg, 61 788, GOx), p-nitrophenyl phosphate disodium salt
substrate to the enzyme active site), manoeuvrability, recycla- hexahydrate (purity $ 99%, 88 485, pNPP), glycine (purity $ 99%,
bility, and additional functionalities for enhancing the overall 52 574), magnesium chloride hexahydrate (purity $ 99%,
performance of the material, would be ideal for the next 69 396), D-glucose (purity $ 97%, 15 405 dextrose) and 2,20-azino-
generation hybrid biomaterials.
bis(3-ethylbenzothiazoline-6-sulphonic acid) (purity $ 98.5%,
Bioconjugation of enzymes with PEG based polymers has 40 157 ABTS) were obtained from SRL chemicals (Mumbai,
been widely used in literature to prepare hybrids with tuneable India). SnakeSkin dialysis tubing (10 kDa cut off, 35 mm dry ID,
interactions of the biomolecule with their environment.15–18 88 245) was bought from Thermo Scientic, USA. NaOH pellets
This synthetic methodology imparts stability to the enzymes and concentrated HCl (35 w/v%) were purchased from Merck
towards denaturation, and can also help in their dispersion in (Mumbai, India). All the chemicals were used as received and
an otherwise non-solvent conditions.18 Further, combining experiments were carried out using MilliQ type I water with
ꢁ
these conjugates with the three dimensional scaffolds made resistivity 18.2 MU cm at 25 C. Regenerated silk broin (RSF)
using nanoparticles can provide a new class of materials with was obtained by degumming of silk cocoons extracted from
desired specications such as high porosity, and handleability Bombyx mori, and subsequently the silk nanoparticles (silk NPs)
for various biological and biocatalytic applications. In this were prepared according to the method mentioned below.
context, ice templating has been recently used to make elastic
and porous scaffolds of nanoparticles and polymers.19,20 These
scaffolds show complete recovery of size and shape even aer
imposing large compressive strains,20 and have been employed
2.2 Preparation of enzyme-polymer surfactant conjugates
(a) Cationization of native enzymes. Cationization of acidic
for various applications such as energy storage devices,21 amino acids (Glu and Asp) on native alkaline phosphatase (nALP)
catalysis,22,23 and tissue engineering.24 However, a facile and surface was carried out via carbodiimide (EDC) mediated
generic method for integrating these scaffolds with different dimethyl-1,3-propanediamine (DMAPA) coupling. The amount of
enzymes, nucleic acids, and other functional biomolecules is DMAPA required was calculated based on the number of acces-
still to be explored. In this work we use protein surface engi- sible acidic amino acid residues on protein surface; DMAPA was
neering to prepare enzyme-polymer surfactant core–shell taken in 1 : 50 molar ratio of accessible acidic residues. Initially,
conjugates and report for the rst time their integration with DMAPA was diluted to 2 M solution in MilliQ water which was then
polyelectrolyte coated regenerated silk broin (silk) or silica neutralized by dropwise addition of 6 N HCl and 0.2 N HCl till pH
nanoparticles to form cross-linked 3D hybrid biocatalytic of the solution became 6.0. Lyophilized nALP powder was dis-
sponges, using ice-templating methodology. We show that, solved in MilliQ water (1 mg mLꢀ1) and added dropwise in DMAPA
whilst the native enzymes either denature or are washed out solution (2 M; pH ¼ 6.1) with constant stirring. Coupling reaction
during the fabrication and usage of the sponges, their core– was initiated with addition of EDC taken in 1 : 25 mole ratio of
shell like enzyme-polymer surfactant conjugate counterparts acidic amino acids residues and added in three steps with 1 hour
are integrated within the sponges. Interestingly, the core–shell interval. Reaction was carried out ꢁfor 24 hours with constant stir-
conjugate based sponges exhibit mechanoresponsive catalytic ring at ambient temperature (25 C); cationized ALP (cALP) was
behaviour with the capability of increasing the rate of subsequently dialyzed using SnakeSkin dialysis tubing (10 kDa
biochemical reaction by simply changing the frequency of MWCO) against MilliQ water which was changed frequently in 48
compression–decompression of the sponge, without much hours to remove excess DMAPA and EDC. Cationization of nGOx
disruption of the enzyme stability. This was attributed to the and nHRP to produce cGOx, and cHRP was done using similar
change in mass transfer and diffusion of the substrate within methodology.
the porous channels of the material on changing the frequency
of compression–decompression.
(b) Synthesis of conjugates using cationized enzymes. An
aqueous solution of polymer surfactant, PS, (1.5 mg mLꢀ1) was
added drop-wise to cALP (0.5 mg mLꢀ1 aqueous solution) with
continuous stirring at 500 rpm. Conjugates were obtained in the
form of clusters that were identied by a marked increment in
opacity of solution which was monitored with Cary 100 UV-
2. Materials and methods
2.1 Materials
Alkaline phosphatase (from bovine intestinal mucosa, lyophi- visible spectrophotometer at 630 nm; maximum opacity i.e.
lized powder, $ 10 DEA units mgꢀ1, P7640, ALP), N,N-dimethyl- formation of conjugates clusters was obtained at protein to
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Fig. 1 (a–d) Figure showing the synthesis of alkaline phosphatase-polymer surfactant bioconjugates (ALP-PS) from native ALP (nALP) and the
corresponding characterization studies involved at each stage of bioconjugation: (a) schematic showing carbodimide (EDC) mediated diamine
(DMAPA) coupling on the nALP surface, with acidic residues highlighted in pink (Asp, Glu), cALP shows post coupling reaction all basic groups
(highlighted in violet) and ALP-PS represents the bioconjugation of cALP after shell formation of PS on cALP by electrostatic interactions. (b)
Dynamic light scattering (DLS) shows decay of autocorrelation function (g2(s,d) ꢀ 1) with respect to delay time (s) in seconds and inset showing
number average distribution in solution for (i) native ALP (nALP; 3.9 ꢂ 0.4 nm), cationised ALP (cALP; 4.5 ꢂ 0.6 nm) and bioconjugate clusters
(ALP-PS; 50 ꢂ 5 nm). (c) Zeta potential measurements on nALP, cALP, and ALP-PS bioconjugate clusters showing a charge on the slipping plane
as ꢀ36.4 ꢂ 1.4, +17.79 ꢂ 0.7, and +2.3 ꢂ 0.1 mV, respectively. This indicates the sign of the charge on the nALP at physiological pH is reversed on
coupling with DMAPA, however on the addition of anionic PS the charge on cALP is neutralized. (d) Circular dichroism spectra for aqueous
solution of nALP, cALP and ALP-PS recorded at room temperature shows a predominant b-sheet structure.
surfactant charge ratio of 0.70. These ALP clusters (ALP-PS) were showed a swelling volume ratio of ꢄ4.9 on complete saturation
characterized by DLS, zeta potential and were used for fabrica- with solvent. This attributed to approximately to 2000 times
tion of biocatalytic sponge as shown in Fig. 1. GOx-PS, and HRP- higher mass uptake then it is original mass. Further, to test the
PS conjugated were also prepared by conjugating cGOx, and enzymatic activity of the sponges, these were equilibrated at
cHRP using similar methodology.
room temperature in glycine buffer (pH 8.8, 100 mM) contain-
ing MgCl2$6H2O (1 mM).
2.3 Fabrication of silica nanoparticles and nALP/ALP-PS
based biocatalytic sponges
2.4 Fabrication of silk nanoparticle and nALP/ALP-PS based
biocatalytic sponges
In a well dispersed 10% w/v solution of silica nanoparticles,
10 mg of 25 kDa PEI (from 100 mg mLꢀ1 stock solution) was This was done in two steps viz. (a) synthesis of silk nanoparticles
added and vortexed for 10 minutes to ensure a layer-by-layer from regenerated silk broin (RSF) extracted from Bombyx mori,
assembly of the positively charged PEI on the negatively and (b) fabrication of biocatalytic sponges from silk nano-
charged surface of silica (Si) nanoparticles. This resulted in particles using similar procedure as explained above for silica.
formation of Si-PEI nanoparticles (NPs) of diameter 45 ꢂ 5 nm Briey silk nanoparticles were synthesized by using desolvation
(Fig. S1d, ESI†). To the resultant colloidal mixture of Si-PEI NPs, method. Typically 5 mL of 50 mg mLꢀ1 aqueous solution of
0.5 mL (0.2 mg mLꢀ1) of nALP or ALP-PS conjugates solution was regenerated silk broin was added drop-wise (ꢄ20 mL) to 20 mL
added, and stirred for 10 min. Finally, 5 mg of polyethylene acetone (maintaining nearly 80% v/v acetone).25 Silk precipi-
diglycidyl ether PEGDE (from 100 mg mLꢀ1 stock solution) was tates out of the solution, and was further collected aer
used to cross-link the nALP/nALP-PS with Si-PEI NPs and frozen centrifuging the solution at 15 000 rpm for 2 hours. The residue
for 24 hours at ꢀ20 ꢁC.20 Aer 24 hours, the sponges were thawed collected aer centrifugation was re-suspended in MilliQ water,
and washed with copious amount of MilliQ water to ensure vortexed and sonicated twice for 30 seconds each time (Fig. S3a
removal of excess PEGDE. These materials were designated as ESI†). The nanoparticles were lyophilized, characterized using
ꢁ
nALP/Si-PEI and ALP-PS/Si-PEI nanoparticle (NP) sponges.
The dimensions of the Si-PEI NPs sponges completely satu-
DLS, zeta potential, and were stored at 4 C for further use.
For fabrication of silk based bio-degradable biocatalytic
rated with 1.5 mL of the buffer/substrate solution, was found to sponge 15 mg of 25 kDa PEI (from 100 mg mLꢀ1 stock solution)
be 1 cm (height) ꢃ 1 cm (diameter). Accordingly, using mate- was added to a well dispersed 1.5 mL aqueous solution of
rials balance a theoretical value of porosity (seen using SEM) (8.5 mg mLꢀ1) silk nanoparticles which was further vortexed
equal to 95.13% was calculated. The completely dried sponges and sonicated for 10 minutes to form a homogenous solution of
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silk-PEI nanoparticles (NPs). To the resultant colloidal mixture concentration range 30 to 450 mM, and 100 mL diluted nALP/
of silk-PEI NPs 0.5 mL of ALP-PS (0.2 mg mLꢀ1 solution) was cALP/ALP-PS solution. Formation of pNP was measured at
added and stirred for 10 min. Finally, 5 mg of PEGDE cross- 37 ꢁC for 15 min with constant stirring, monitored continuously
linker (from 100 mg mLꢀ1 stock solution) was added and the at 410 nm using UV-visible spectrophotometer. The initial rate
ꢁ
mixture and frozen for 24 hours at ꢀ20 C. Aer 24 hours, the of reaction was calculated and plotted against substrate
sponge was thawed and washed with copious amount of MilliQ concentration to obtain Michaelis–Menten kinetic plot which
water to ensure removal of excess PEGDE; sponge was then was then tted using Michaelis–Menten function provided in
equilibrated with glycine buffer (pH 8.8, 100 mM) containing Origin soware (Fig. 4).
MgCl2$6H2O (1 mM). The sponges were designated as nALP/
silk-PEI or ALP-PS/silk-PEI nanoparticle (NP) sponges.
To determine the hydrolytic capacity of immobilized nALP/
ALP-PS within the catalytic sponges, small cylindrical sponges
(total sponge swollen volume ¼ 0.7 mL; containing 0.25 mg
total protein) were used. These sponges were equilibrated to
ambient temperature prior to assay. For these experiments at
2.5 Michaelis Menten kinetic studies
ꢁ
Enzymatic activity of all samples was determined via steady
state Michaelis–Menten kinetic studies. The measurements
were performed with increasing concentrations of substrate
solution, and continuous monitoring of product formation was
done with respect to time. Subsequently, the amount of product
formed per minute (velocity of reaction) was calculated from the
slope of the curve. Product formation was quantied using Cary
100 UV-visible spectrophotometer equipped with temperature
controller dual cell Peltier unit. All aqueous samples of nALP,
cALP, and ALP-PS conjugate clusters, were diluted to protein
concentration of 0.1 mg mLꢀ1 in MilliQ water before adding to
assay mixture. Reaction mixture comprised of 1.5 mL glycine
37 C, the sponge was suspended in the reaction cuvette with
the help of an inert thread, and the substrate solution was
stirred continuously at 200 rpm by using rice size magnetic
needle to ensure complete mixing of all components and to
avoid diffusion limitations. Kinetic mode in the UV-Vis spec-
trophotometer was used to measure the absorbance at 410 nm
continuously for 15 minutes (detailed explanation regarding
calculations for different kinetic parameters are explained in
ESI†).
2.6 ON–OFF switching using biocatalytic sponges
buffer (100 mM, pH 8.8 containing 1 mM MgCl2$6H2O), 1.5 mL ON–OFF switching studies were carried out for ALP sponges in
substrate solution (p-nitrophenyl phosphate; pNPP) with UV cuvette (3 mL); reaction mixture comprised of glycine buffer
Fig. 2 Schematic illustration of fabrication of biocatalytic sponges using ice templating methodology: an aqueous solution of well dispersed
polyethyleneimine (PEI) coated silk (silk-PEI NPs) or silica nanoparticles (Si-PEI NPs) mixed with either (a) native enzymes (nALP/nGOx/nHRP) or
(b) enzyme-polymer surfactant core–shell bioconjugate (ALP-PS/GOx-PS/HRP-PS) was frozen at ꢀ20 ꢁC for 24 hours in the presence of
polyethylene diglycidyl ether (PEGDE) as a cross-linker. In this schematic, the sponge made using nALP (a) and ALP-PS (b) have been shown. This
results in highly porous (as seen from SEM images) biocatalytics sponge made of three dimensional connected network of nanoparticles (NP) and
native enzyme/enzyme-PS walls. Snapshot images shows nALP/Si-PEI (centre, top) and ALP-PS/Si-PEI (centre, bottom) sponges and indicates
the lack of structural strength in nALP biosponge in comparison to ALP-PS immobilized Si-PEI NPs biosponges. The mechanical differences arise
due to relatively lesser number of basic residues on the surface of nALP as compared to ALP-PS. This results in poorer crosslinking with PEGDE.
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100 mM, pH 8.8 containing 1 mM MgCl2$6H2O and substrate and 25 minutes. Control experiment was carried out by allowing
solution 1000 mM mixed in 1 : 1 volume ratio with total volume substrate conversion without compressing sponge (static mode)
3 mL. Sponges were equilibrated at room temperature prior to for similar time periods; all studies were carried out in tripli-
use. Sponge was immersed in solution using thread and cates and standard deviation was calculated for each data point.
formation of product (p-nitrophenol) was measured at 37 ꢁC,
410 nm for 5 min (ON mode). Under OFF mode, sponge was
removed and change in absorbance was monitored at 37 ꢁC,
3. Results and discussion
410 nm for 5 min. Alternate cycles of ON–OFF mode were per- The biocatalytic scaffold-like spongy material comprising
formed 6 times.
nanoparticles and enzymes was prepared by using an ice-
templated methodology as described in the literature.18,19 In
a modied procedure, polyethyleneimine (PEI; Mw ¼ 25 kDa)
coated regenerated silk nanoparticles (silk NPs; hydrodynamic
diameter; Dh ꢄ106 ꢂ 10 nm; Fig. S1 and S3, ESI†) or silica
nanoparticles (Si-NPs; Dh ꢄ25 ꢂ 2 nm; Fig. S1, ESI†) were mixed
with either native alkaline phosphatase (nALP; homodimeric
2.7 Reusability (recyclability) studies
To test the durability of biocatalytic ALP-PS/Si-PEI sponges, we
performed multiple enzymatic hydrolysis cycles on a single
sponge using a reaction mixture that contained 1.5 mL glycine
buffer (100 mM, pH 8.8 containing 1 mM MgCl2$6H2O); 1.5 mL
2 mM substrate solution (p-nitrophenyl phosphate, pNPP).
Reusability study comprised of 10 compression–decompression
(C–D) cycles carried out at 1 C–D cycle per min. At the end of
each cycle the product was squeezed out and the sponge was
washed with copious amounts of 50 mM NaCl solution and
water. Similar reusability studies were performed on sponges
containing nALP and cALP.
dephosphorylating enzyme, Mw
¼
86 kDa), or alkaline
phosphatase-polymer surfactant core–shell conjugates (ALP-PS;
Fig. 1a); ALP-PS conjugates were pre-synthesized by surface
engineering of the nALP with poly(ethylene glycol) 4-nonyl
phenyl 3-sulfopropyl ether potassium salt (PS; Mn ¼ 1200). The
resultant mixtures (molar ratio of silk-PEI : nALP or ALP-PS and
Si-PEI : nALP or ALP-PS was kept at 5.3 ꢃ 10ꢀ4 : 1 and 4.2 : 1,
respectively) were then kept in the freezer at ꢀ20 ꢁC, and
allowed to cross-link slowly by using polyethylene diglycidyl
ether (Fig. 2). Aer 24 hours the snow white sponges were taken
out of the freezer, thawed and investigated further for their
mechanical and biocatalytic property. As a result of ice tem-
plating, both the sponges showed high level of porosity as seen
by SEM (Fig. 2). However, visual observation depicted that
sponges comprising nALP/Si-PEI or nALP/silk-PEI exhibited
poor mechanical strength, whilst the ALP-PS/Si-PEI or ALP-PS/
silk-PEI nanoparticle sponges were more sturdy, and robust
2.8 Compression–decompression (C–D) studies
The sponges could be compressed instantaneously to approxi-
mately 40% of its initial height by applying an effective stress of
approximately 22.9 kPa. The effective stress can be calculated
using the following equation.
Effective stress ¼ [m ꢃ g/(A ꢃ (1 ꢀ p))];
where, m ¼ (35.7 g) mass is that which is required to compress (Fig. 1, centre). This indicated that the ALP-PS conjugates
the sponge to 40% of its initial height, g ¼ (9.8 m sꢀ2) gravita- integrated effectively within the nanoparticle-PEI sponges, and
tional acceleration, A ¼ (p ꢃ r2) cross-sectional area of the were cross-linked efficiently. This could be understood by the
sponge with radius, r (0.01 m), p ¼ (95.13%) porosity.
DLS observation (Fig. S1d†), which showed formation of micron
On removal of the stress the sponges regained its original size aggregates (z1.5 mm; Fig. S1c and d, ESI†), perhaps due to
shape and size. This property of the catalytic sponges was used electrostatic attraction between nALP (zeta potential z ꢀ36.4 ꢂ
for studying any change in the rate of enzymatic conversion of 1.4 mV; Fig. 1) and Si-PEI NPs (zeta potential z +17.0 ꢂ 0.2 mV;
the substrate by employing different compression–decompres- Fig. S1a and b, ESI†); visual observation also showed sedimen-
sion (C–D) cycles per minute. A 1.5 mL (saturation level for 1 cm tation of these aggregates from the solution mixture. On the
ꢃ 1 cm sponge) of buffered substrate solution containing pNPP contrary, mixing of ALP-PS with silica-PEI NPs did not lead to
was pipetted on the ALP-PS/Si-PEI NP biocatalytic sponge. The any large aggregates and the mixture was homogenous and
enzymatic conversion of p-nitro phenyl phosphate (pNPP) to p- stable upto 48 h of observation. It should also be noted that the
nitro phenolate ion (pNP) was carried out for 5 minutes at 25 ꢁC increase in the number of cross-linkable amine groups gener-
with intermittent compression (and subsequent immediate ated on the protein surface during bioconjugation step (nALP ¼
decompression) of the sponge aer every 1 minute. At the end of 41; ALP-PS ¼ 79; Fig. S2 ESI,† Fig. 1a) could be attributed
5 minutes, the product was squeezed/pressed out by applying towards efficient crosslinking and formation of sturdy ALP-PS
pressure and sponge was successively washed with 200 mM based biocatalytic sponges.
NaCl solution in order to ensure complete product recovery. The
Further, both the Si-PEI NP sponges (nALP and ALP-PS
extruded solution was then analyzed for concentration of p- based) were tested for their enzymatic activity at 25 ꢁC by
nitrophenol (produced in 5 minutes of reaction time and 1 C–D pouring 1.5 mL (saturating volume for sponges) of substrate p-
cycle per minute) using Cary 100 UV-visible spectrophotometer nitrophenyl phosphate (pNPP; 15.2 mM in 100 mM glycine
by measuring absorbance at 410 nm. Similar studies for 1 C–D buffer with 1 mM MgCl2, pH 8.8), and monitoring the dephos-
cycle per min were done for 10 min, 15 min, 20 min and 25 min phorylated product p-nitrophenolate ion (Fig. 3a and S4 ESI†).
incubation (each study in separate sponge). Further, reaction There was a visually noticeable color change of the sponges
rates were also determined at 2 and 4 C–D minꢀ1 for 5, 10, 15, 20 from colorless to bright yellow within rst 5 minutes which
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PS sponge showed only the presence of 410 nm peak, indicating
the formation of pNP, the spectra of solution from nALP sponge
had an additional peak at 280 nm, suggesting leaching of the
native protein (Fig. 3c). It was also observed that the structural
integrity of the ALP-PS sponge was totally preserved aer
squeezing out the product, whereas the nALP sponges were
highly disintegrated (Fig. S6, ESI†).
Quantitative estimation for the activity of ALP in either in the
aqueous solution of nALP, cALP, and ALP-PS, or sponges of ALP-
PS was done through Michaelis–Menten kinetics (Fig. 4). These
studies indicated that the kcat of nALP (in the aqueous solution)
decreased by approximately 35% aer cationization to form
cALP (from z20 500 minꢀ1 to z13 500 minꢀ1), however,
remained almost similar to cALP upon conjugation with poly-
mer surfactant (ALP-PS; approx. 14 000 minꢀ1; Table 1).
Further, the immobilization of ALP-PS in the biocatalytic
sponge led to z5 time further decrease in the kcat value
(compare data for ALP-PS aqueous solution to ALP-PS in the
sponges Table 1). This was also accompanied by an increase in
Km from 45 mM to 606 mM, which could be understood as
a result of diffusion limitation of the substrate in the porous
sponge.
Having established a marked difference between nALP and
ALP-PS immobilized sponges towards their mechanical and
catalytic properties, we further tried to investigate the possi-
bility of reuse of single sponge over multiple catalytic cycles.
One of the important property of these sponges is their ability to
undergo compression–decompression (C–D) cycles without any
structural degradation (Fig. 5a and b).19,20 The sponges could be
compressed instantaneously to approximately 40% of its initial
height by applying an effective stress of 22.9 kPa, without any
structural deterioration (Fig. 5a). This inherent elastic property
was utilized to carry out the recyclability experiments (Fig. 5c,
inset). These studies were done for two different sponges of Si-
PEI NPs containing 0.2 mg ALP either in the form of nALP or
ALP-PS core–shell conjugates. A 1.5 mL aqueous solution con-
taining 2 mM pNPP (Fig. 5c, inset) was poured on both the
sponges (initially white in colour) and the reaction was allowed
to occur at 25 ꢁC for 10 minutes with intermittent C–D aer
every 1 minute to ensure proper mixing. Aer the completion of
the reaction, the product, pNP, was extruded out by applying
effective normal stress. However the colour of both the sponges
still remained yellowish, which was a result of residual pNP
which could not be extruded. This was attributed to electrostatic
interaction of anionic pNP and the cationic PEI of the sponge.
To alleviate this problem the sponges were soaked in 50 mM
NaCl solution for 2 minutes before washing with MilliQ water
and reusing the sponge for next cycle (Fig. 5c, inset). It was
observed that nALP immobilized sponges lost approximately
entire activity within three reusability cycles, however, the
activity of ALP-PS decreased by only ꢄ10% over similar number
of recycles. Aer 10 recycles the activity of ALP-PS sponge
decreased only by ꢄ33% (Fig. 5c). This indicated signicant
advancement when compared to native ALP immobilized
sponges, and suggested that polymer surfactant shell
surrounding the ALP enzyme (in ALP-PS) not only imparted
stability to the biomolecule towards denaturation, but also
Fig. 3 Hydrolysis of p-nitro phenyl phosphate (pNPP) to p-nitro
phenolate ion (pNP) occurring within the biocatalytic silica-PEI NPs
sponges: (a) enzymatic reaction of conversion of pNPP (Absmax at 305
nm) to pNP (Absmax at 410 nm) using ALP/ALP-PS immobilized
sponges. (b) Snapshot images showing time dependent (0 to 25
minutes) enzymatic reaction in ALP-PS cylindrical sponge (2 cm ꢃ
1 cm; diameter ꢃ height) at 25 ꢁC; scale bar 1 cm. The top panel in (b)
shows pipetting 1.5 mL substrate solution comprising 15.2 mM pNPP
on the sponge. Progress of reaction is visualized by the change in color
of the sponge from snow white to bright yellow. (c) Corresponding to
snapshot images, the UV-Vis absorbance spectra of ALP-PS sponge at
different time intervals showing a decrease in concentration of the
substrate, pNPP, at 305 nm and a simultaneous increase in the
concentration of product, pNP, observed at 410 nm inside the sponge.
The dashed circle shows absorbance at 280 nm from the extrudate of
nALP based sponge obtained after 25 minutes. This indicated the
leaching of the nALP enzyme from the sponge. The inset in (c) shows
the application of stress using flat spatula for extruding the product out
of the sponge and collection in an Eppendorf tube.
grew intense over 25 minutes (Fig. 3b and S5, ESI†). Control
experiments without the ALP/ALP-PS in the sponges did not
result in color change, indicating the enzymes were responsible
for the dephosphorylation reaction and the associated colour
change (Fig. S5 ESI†). As these sponges were highly compress-
ible and regained shape-size aer removing the compressive
stress,19 the product could be extruded out by applying
a mechanical pressure using a at spatula (Fig. 3c, inset), and
subsequently monitored using UV-Vis spectrophotometer. It
was observed that the color change was in fact associated with
the dephosphorylation of pNPP to form p-nitro phenolate ion
(pNP) (correlating the activity of immobilized core–shell ALP-PS
based conjugates) which absorbed at 410 nm (Fig. 3c). Inter-
estingly, it was also observed that whilst the extrudate from ALP-
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changing the frequency of C–D cycles, over the 25 minutes of
observation, it was that the onset for the pNP production
occurred faster for biocatalytic sponges subjected to higher C–D
frequency. This could be observed from the slopes of the curves
obtained for pNP concentration vs. time (Fig. 5d). A highest
slope (indicating rate of pNP formation) of ꢄ12.8 (for 4 C–D
cycles per min) was observed in between 10–15 minutes (total
40–60 cycles), while the highest slopes of ꢄ10.9 (for 2 C–D cycles
per min), and ꢄ8.3 (for 1 C–D cycle per min), were observed
between 15–20 minutes (total 30–40 cycles), and 20–25 minutes
(total 20–25 C–D cycles), respectively. Without employing any
C–D cycle, the rate of formation of product was very sluggish,
and the amount of pNP formed over 25 minutes (24.52 ꢂ 4.43
mM) was approximately 8 times less than that formed using 4
C–D cycles per min (190 ꢂ 9.21 mM). In a control experiment
performed with pNPP as the substrate solution and subjected to
4 C–D cycles per min with sponges devoid of nALP/ALP-PS, no
formation of pNP was observed suggesting that the enzymatic
reaction occurred only due to ALP-PS, but the rate could be
controlled using C–D property of the sponge.
The increase in substrate conversion with increasing C–D
cycle frequency suggested that the reaction could be limited by
mass transport within the pores. In a simplistic assumption our
system can be treated as an analogue to a porous solid catalyst –
uid reactant system, where the sponge can be thought of as
a porous catalyst with immobilized enzyme molecules as the
active sites of the catalyst.26 Such systems are affected by mass
transfer limitations.27 We hypothesize that on decompression,
the convective transport of pNPP within the sponge pores is
increased. On applying compressive stress, the pores shrink,
forcing the reaction solution out of the sponge, thereby
enhancing transport of mixture of pNPP and pNP out of the
pores. It was also interesting to note that similar studies con-
ducted with a biocatalytic sponges based on ALP enzyme mimic
ceria nanoparticles (instead of a combination of ALP-PS and Si-
PEI nanoparticles; unpublished results), indicated a similar
increase in the product formation on increasing C–D cycle
frequency. This suggested that the variation in the rate of
formation of pNP was in fact related to C–D frequency, however
any change in the secondary structure of the enzyme as a result
of the C–D cycles, and its effect on the accessibility of the active
site by the substrate cannot be actually ruled out.
Fig.
4 Michaelis–Menten kinetic studies: comparative Michaelis–
Menten kinetics studies for aqueous solutions of nALP, cALP, ALP-PS,
and ALP-PS in the biocatalytic silica NPs-PEI sponge. Reaction mixture
comprised of 1.5 mL glycine buffer (100 mM, pH 8.8 containing 1 mM
MgCl2$6H2O); 1.5 mL substrate solution (paranitrophenyl phosphate,
pNPP) with concentration range 30 to 450 mM and 100 mL, 0.1 mg
mLꢀ1 diluted ALP solution for nALP, cALP, ALP-PS. Small cylindrical
sponges were used for assaying ALP-PS activity (0.7 mL volume
containing 0.25 mg total protein), these sponges were equilibrated to
ambient temperature prior to assay. Snapshots bottom right inset,
show cylindrical sponges before and after reaction suspended in the
cuvette with the help of a thread. Enzyme assay was carried out at
37 ꢁC for 15 min with constant stirring, monitored continuously at
410 nm using UV-visible spectrophotometer.
prevented leaching (through efficient crosslinking) during
repeated usage. Given the disadvantages associated with the use
of nALP, we decided to further work only with ALP-PS/Si-PEI
based sponges.
Interestingly, it was observed that the elasticity driven
compression–decompression (C–D) of the sponges could be
utilized for changing the rate of enzymatic conversion of pNPP
to pNP. The frequency of compression-decompression (C–D)
was varied to enhance the product formation as shown in
Fig. 5d. The compression of the ALP-PS/Si-PEI sponges was
accompanied by the extrusion of the reactant (15.2 mM pNPP)/
product (pNP) mixture solution which could be collected at
different time periods for UV-Vis measurements (similar to
Fig. 3c, inset). Aer a wait of 2 seconds in the compressed state,
the stress was removed and the sponge was allowed to decom-
press under ambient conditions. This lead to the absorption of
the extrudate back in the sponge. The entire C–D cycle was
completed (sponge regaining its original shape on removal of
effective compressive stress) within 4 to 5 seconds. Upon
To explore other properties of this class of biomaterial we
employed the ALP-PS/Si-PEI NP sponges for the ALP based
dephosphorylation reaction that can be switched ON (activated)
or OFF (deactivated) by the mechanical manoeuvring of the
sponges in (immersing) and out (withdrawal) of the pNPP
Table 1 Enzymatic parameters obtained from Michaelis–Menten kinetic fits to the enzymatic assay data shown in Fig. 4
Parameters
nALP (aq.) ꢂ std. dev.
cALP (aq.) ꢂ std. dev.
ALP-PS (aq.) ꢂ std. dev.
ALP-PS in sponge
Vmax (mM minꢀ1
)
3.59 ꢂ 0.29
359.0 ꢂ 29
2.35 ꢂ 0.17
235.7 ꢂ 17
2.46 ꢂ 0.11
246.5 ꢂ 11
4.11
51.8
606.2
2962.5
4.9
Vmax (mM minꢀ1 mgꢀ1
Km (mM)
)
119.7 ꢂ 15.8
20 502.5 ꢂ 1656
171.3 ꢂ 25
41.3 ꢂ 0.62
41.5 ꢂ 13.8
kcat (minꢀ1
)
13 460.8 ꢂ 971
325.9 ꢂ 19.3
14 077.6 ꢂ 605
338.9 ꢂ 147
ꢀ1
k
cat Km (mMꢀ1 minꢀ1
)
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Fig. 5 Utilizing the elastic property of sponges for recyclability and enhancing rate of reaction: (a and b) optical images showing compression
and decompression (C–D) of (1 cm ꢃ 1 cm) the Si-PEI NPs sponge immobilized with either nALP or ALP-PS with the help of a mechanical stage.
The sponge regains its original size and shape from to 40% of its initial height after removal of compressive stress (decompression). (c) Multiple
reaction cycles were performed on a single sponge (containing 0.2 mg ALP-PS) using 2 mM pNPP at room temperature. Reusability cycles using
nALP (green, open circles) and ALP-PS sponges (black, open circles). A typical reusability cycle consists of addition of substrate to the sponge,
performing 1 C–D cycle per min for 10 minutes, extruding the product, washing the sponge first with 50 mM NaCl then with MilliQ water and
reusing the sponge for next cycle (inset in panel (c)). (d) Graph depicting dependence of compression and decompression rate on the formation
of pNP. Three different sets of C–D frequencies viz. 1, 2, 4 minꢀ1 were applied and [pNP] was measured by absorption spectroscopy at 410 nm at
different time intervals of 5, 10, 15, 20 and 25 minutes. Inset in (d) clearly demonstrates that [pNP] is approximately 3 higher within the first 5
minutes when 4 C–D cycles per min are employed.
substrate solution, respectively. Time dependent ON–OFF cycles, with each activation providing approximately similar
switching was activated by the immersion of ALP-PS based rate of formation of pNP over 5 min, whereas the deactivation
sponge in a 3 mL solution (1 mM pNPP; 1 5 mL, 100 mM glycine (OFF) cycle showed no formation of pNP (Fig. 6 and S7, ESI†).
buffer (pH 8.8) with 1 mM MgCl2; 1.5 mL) for 5 minutes, and Overall these experiments suggested that the protein-polymer
increase in absorbance related to the formation of pNP was surfactant conjugate based catalytic sponges were structurally
measured at 410 nm (ON-1; Fig. 6a). Aer 5 minutes of stable and could be utilized for temporal control of the
immersion the switch was deactivated by withdrawing the biochemical reactions whilst simultaneously allowing for
sponge out of the solution. The absorbance of the substrate effortless recovery of the material conning the enzymes.
solution in the cuvette did not change during the 5 minute
Aer establishing the use of biocatalytic sponges for a single
deactivated period (OFF-1), suggesting again a minimal leach- enzyme based biochemical transformation, as a proof of
ing of the enzyme in the solution (Fig. 6b). However, as the concept, we chose to utilize them for driving GOx and HRP
sponge was re-immersed in the solution for step ON-2, during based cascade reaction involving conversion of D-glucose to
the initial 2 minutes of switch activation a steep increase in gluconic acid and H2O2, followed by oxidation of the later in the
formation of pNP occurred aer which the rate of reaction presence of reducing dye molecule (Fig. 7a). The GOx-PS + HRP-
slowed down (as indicated by two different slopes in Fig. 6b, PS/Si-PEI nanoparticle based ice-templated sponges were
bottom inset). This suggested that the reaction continued in fabricated in a similar way as ALP-PS (Methods, ESI†). The dual
residual substrate solution contained in the sponge even in the enzyme-PS sponges were mechanically robust and showed
deactivated period. It was attributed to the fact that these catalytic activity (Fig. S8, ESI†). The cascade reaction was acti-
sponges behaved like hydrogels,19 and it is intuitive that some vated by immersing the GOx-PS + HRP-PS and Si-PEI sponge in
aqueous solution will always be retained in the sponge even an aqueous substrate solution comprising D-glucose and an
aer their removal from the cuvette. A similar activation (ON)/ electron
donor
(2,20-azino-di-(3-ethylbenzothiazoline)-6-
deactivation (OFF) behaviour was observed for the next six sulfonic acid (ABTS). It was observed that the colour of the
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product ABTS+c (Fig. 7c). Control experiments performed with
sponge being withdrawn from the substrate solution showed no
increase in absorbance at 414 nm, suggesting no leaching of the
enzymes involved in the cascade.
4. Conclusions
In conclusion, we have shown the development of enzymatically
active biocatalytic sponges fabricated using ice templating of an
aqueous mixture of silica (inorganic) or silk (biodegradable)
nanoparticles and core–shell enzyme-polymer surfactant
conjugates. Signicantly, we show that while the native enzymes
tend to form sponges that structurally disintegrate on applying
very few consecutive mechanical stresses with accompanying
rapid loss in activity, their enzyme-polymer surfactant coun-
terparts immobilize efficiently to form sturdy, robust, highly
catalytic, and recyclable sponges. These core–shell conjugate
based catalytic sponges exhibit mechanoresponsive and recy-
clable catalytic behaviour with the capability of tuning the rate
of reaction by changing the frequency of compression–decom-
pression of the sponge. This can be attributed to the change in
mass transfer and diffusion of the substrate within the porous
channels. This work, therefore, represents development of new
class of biocatalytic material, which can nd application in
remediation of polluted water, whereby the toxic pollutant
(along with water) can be soaked in the biosponge, and then
allowed to be catalytically converted into non-toxic product,
which can be later squeezed out. We also envisage that these
sponges can be used for efficient and economical biochemical
transformations in application related to tissue engineering,
drug delivery, biocatalysis, and biosensing.
Fig. 6 ON–OFF switching of the reaction using ALP-PS and silica-PEI
sponges: (a) snapshots of cuvette with the ALP-PS/Si-PEI NP based
sponge either immersed (ON) or removed out (OFF) from the pNPP
substrate solution to demonstrate an enzymatic switch for controlling
the reaction. (b) Time dependent UV-Vis spectra for the ON–OFF
switch of ALP-PS sponge cycled 6 times in the presence of pNPP
solution, with complete set (ON; red circles and OFF; blue circles)
lasting for 10 minutes at 37 ꢁC. Snapshots in top inset, (b) show the
reaction set up with the ON and OFF steps represented by ALP-PS
sponge immersed and withdrawn from the reaction mixture, respec-
tively. The bottom inset, (b) shows the normalized absorbance at
410 nm for ON–OFF set-2 suggesting two different rate of formation
of pNP indicated by two different slopes.
entire substrate solution turned light green within couple of
minutes, and became much intense over 10 minutes (Fig. 7b).
The reaction could also be monitored by measuring the absor-
bance at 414 nm associated with the production of oxidised
Conflicts of interest
There are no conicts to declare.
Acknowledgements
K. P. S. thanks SERB-DST for funding through grant number
EMR/2016/000174 and IRCC, IIT Bombay for seed grant number
15IRCCSG029. We thank Rajkumar from Department of
Chemistry, IITB for specically synthesizing silk nanoparticles
for our studies.
Notes and references
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