Bioorthogonal in Situ Hydrogels Based on Polyether Polyols for New

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Bioorthogonal in situ Hydrogels based on PolyetherPolyols for new Biosensor Materials with high Sensitivity Anna Lydia Herrmann, Lena Kaufmann, Pradip Dey, Rainer Haag, and Uwe Schedler ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01860 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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ACS Applied Materials & Interfaces

Bioorthogonal in situ Hydrogels based on Polyether-Polyols for new Biosensor Materials with high Sensitivity Anna Herrmann1#, Lena Kaufmann1#, Pradip Dey1, Rainer Haag1, Uwe Schedler1,2*

1

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3, Berlin 14195,

Germany 2

PolyAn GmbH, Rudolf-Baschant-Straße 2, 13086 Berlin

Correspondence: [email protected] (U.S.) #

These authors contributed equally.

Abstract Both non-covalent and covalent encapsulation of active biomolecules, e.g. proteins and oligonucleotides for a new biosensor matrix in an in situ bioorthogonal hydrogel formation via a strain promoted azide-alkyne cycloaddition (SPAAC reaction) was investigated. Unspecific interaction between the gel and the biomolecules as well as protein denaturation was prevented by the bioorthogonal gel components which ensure a uniform aqueous environment in the hydrogel network. No leaching of the active biomolecule was observed. Additionally, a much higher and also adjustable loading of biomolecules in the hydrogel matrix was achieved compared to conventional biosensor surfaces where the sensor molecules are immobilized on monolayers (2D surfaces) or brush-like structures (3D surfaces). Spotting experiments of the hydrogel confirm the possibility to use this new surface for microarray based multiplex applications which require very high signal-to-noise ratios.

Keywords Biosensors, 3D matrix, surfaces, immunoassays, hybridization, bioorthogonal reaction, click reaction, in situ hydrogel

ACS Paragon Plus Environment

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1 Introduction Developing more sensitive and specific biosensors remains a great challenge since the specific verification of analytes plays a major role in biomedical analysis. Immobilizing biomolecules on surfaces typically occurs by adsorptive forces like electrostatic interactions, H-bonding, and dispersive interactions. Molecules can even be immobilized this way on unmodified surfaces of many different materials like polymers, glass, or metal oxides, but it is quite often difficult to control the molecules due to their limited stability and low coverage.1-3 The proximity of the biomolecules to the surface as well as the adsorption process itself can influence and change its structure or lower its activity.2,

4-6

An alternative approach is to

covalently link a biomolecule to a functionalized surface, which is better controllable and provides more stability.5 The capture biomolecule (probe) can be immobilized on different kinds of surfaces (substrates). They can be differentiated between 2D and 3D surfaces.7 The first one is typically built up by alkanethiol self-assembled monolayers (SAMs) on gold or silane-modified layers on silica substrates.8 To overcome the limited loading capacities of monolayers brush-like polymers are attached to the surface by plasma treatment or by covering the surface with covalently anchored polymers like dendrimers to create a 3D surface. They exhibit a high density of functional groups, which can raise the number of functional groups up to 50 per nm2.9-15 Dendrimers and hyperbranched polymers, such as polyamides, represent a class of structurally controlled macromolecules that are used to immobilize a wide range of biomolecules.16-17 Since the macromolecules tend to be very densely packed and the biomolecule may even be bigger, only a few functional groups can be addressed with the consequence that most of them remain unreacted.18-19 When streptavidin is immobilized on a polyacrylic acid surface, only 0.1% of the carboxylic groups can be addressed. By using copolymers, this can only be increased to 0.5%.20 Additionally, it is a huge challenge to avoid an unspecific interaction between the biomolecule and the surface, which is often accompanied with an inaccessibility or loss of the specific binding site of the biomolecule.21 One approach to insert more distance between the substrate and the probe, is to insert a linker that bridges the functionalized surface with the biomolecule.5, 22 Many biomolecules are very delicate not only because covalent or non-covalent interactions can influence their binding sites, but also the environment affects the molecules.23-24 By the inclusion into polymeric gels, biomolecules can be prevented from diffusing out of the matrix, while smaller molecules, such as the target or effector molecules, easily diffuse. The gel entrapment or encapsulation is generally gentle compared to molecules covalently bound to the solid matrix. The most common matrices are alginate, collagen, cellulose, other ACS Paragon Plus Environment

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polysaccharides, polyacrylamide, gelatin, polyvinyl alcohol (PVA), silicones, or pre-polymers (e.g., polyurethanes), which are crosslinked by adding water or by UV irradiation. For some applications sol-gel processes forming silica materials are used.25-26 An example of a polysaccharide is the matrix of carboxymethylated dextran, a flexible unbranched carbohydrate polymer. Immobilized on a gold surface that is an approximately 100 nm thick layer, this polymer is used on most sensor chips for surface plasmon resonance (SPR). The dextran matrix is equivalent in concentration to an aqueous solution of about 2% dextran.27-29 Ossipov et al. and Gacal et al. demonstrated a possibility for the synthesis of hydrogels based on PVA via Click-Chemistry. The degree of modification is only in the range of 1 to 5% to prevent loss of water solubility.30-31Thus, hydrogels like agarose gels or polyacrylamide gels have been used to mimic the physiologic environment together with an increase of the loading capacity, since they are cross-linked networks that retain a large amount of water in their swollen state but still maintain their three-dimensional structure.32-34 Their high water content and soft nature along with their porous structure provide ideal conditions for accommodating biomolecules like active proteins and peptides.35-37 New polymeric networks with an easy and fast cross-linking chemistry that does not affect the original activity of the biomolecules have been intensively investigated, especially those based on dendritic polyglycerol (dPG) and polyethylenglycol (PEG) polymers.35,

38-42

Both polymers are known for their anti-fouling

properties and they prevent nonspecific binding.43-45 Large diffusion barriers, along with the extensive incubation times until the thermodynamic equilibrium is reached, still represent a major challenge. The molecules must be prevented from diffusing out, for which a covalent or non-covalent interaction with the hydrogel is required, which in turn can affect the biological activity.33, 46-49



Figure 1. Principle of the in situ hydrogel that already encapsulates biomolecules during the gel formation. Furthermore, it can act as pre-filter by excluding larger biological entities. For better clarity, a 2D representation of the 3-dimensional network is depicted.

Here, we present a novel surface-bound in situ hydrogel for highly sensitive biosensors (Figure 1). In this new approach, the biomolecule is already present, while the gel is formed from two different building blocks, dPG (a multivalent macromolecular crosslinker) and PEG (a linear macromolecular spacer/crosslinker). It encapsulates the biomolecule upon crosslinking. This not only ensures a high loading with enhanced sensitivity due to the 3dimensional structure, but the premixing also entails in an optimal distribution of the testing probe without any diffusion limitation. In case of a non-covalent entrapment, an optimally adjusted mesh size prevents the sensor molecules from leaching and larger biological entities, e.g., antibodies, viruses, and cells can be excluded. However, if the probe molecule is as small as the diffusing target species, the sensor molecule can also be immobilized covalently using the in situ approach. This bioorthogonal hydrogel, which is built up very fast and carries optional groups for the specific immobilization of smaller molecules, is an approach to master the universal demands of biosensor carrier matrices.


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2 Results and Discussion 2.1 Components and principle performance Concerning the design of the gel components several requirements were considered: First, the gel should be built up from two components, hubs and connectors, which react with each other at room temperature, in water, and without the need of any catalyst. Second, the gelation should be bioorthogonal to avoid any interaction with sensitive biomolecules. Last, the mesh size should be adjustable to ensure an optimal fit for the encapsulated biomolecules. To fulfil all these requirements, we chose dendritic polyglycerol (dPG) functionalized with azide groups for the hubs and polyethylenglycol (PEG) chains containing two cyclooctyne units for the connectors (cf. Scheme 1 A). By changing the length of the PEG the mesh size of the gel can be tuned. The strain-promoted azide-alkyne cycloaddition (SPAAC) between an azide and cyclooctyne is known to be very fast and high yielding without any by-products (cf. Scheme 1 B). A 3D network is formed, where the distance between the hubs is determined by the respective use of PEG linkers, which also account for a certain degree of flexibility.

Scheme 1. (A) The gel consists of two components: a dendritic polyglycerol functionalized with azide groups and a polyethylenglycol chain (MW 3000 or 6000 Da) containing two cyclooctyne units. (B) The crosslinking of both components occurs via strain-promoted azide-alkyne cycloaddition. (C) Schematic presentation of the ideal network with (D) azide silane-functionalized surfaces to anchor the hydrogel covalently.

Mixing both components, the modified dPG and PEG, in a ratio of 1:3 in water, the gel formation occurred within minutes, depending on the concentration, which led to a stable hydrogel. We used glass slides or silica wafers to immobilize the gel on a surface. They were activated with piranha solution or sodium hydroxide solution and treated with a hetero bifunctional azido-PEG-silane that generates a surface with free azide groups to anchor the gel covalently (cf. Scheme 1 D).



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After testing several coating methods spincoating and spotting were identified to yield the best results. The hydrogels with respect to the preparation method and their composition are listed in Table 1. In case of spincoating, both components were mixed by shaking 30 seconds and after a certain period of waiting time the mixture was brought onto the slide and spincoated (2 minutes 150 rpm followed by 1 minute 1500 rpm). Depending on the incubation time which Table 1. Description and composition of the dPG-PEG hydrogels for the respective preparation methods. dPG is always 1 kDa dPG with 20% N3 groups. M %w

V

V n (BCN)

(PEG) (PEG)

[µL]

(dPG) [mmol]

n(BCN)

%w (dPG)

[kDa]

n (N3)

n(PEG)

%w

n(dPG)

(Gel)

(additi n(N3)

[µL]

V

onal)

Spin-

5

100

3

3.33

20

3

1.62

2

-

3

5.4

coating

5

100

6

1.67

20

1.5

0.81

2

-

3

5.2

5

10

3

0.33

20

0.3

0.16

2

-

3

5.4

1

17

6

0.06

2

0.5

0.03

2

10

3

0.5

Handspotting Spotting gel sol

was between 0 and 10 minutes, a layer thickness between 100 nm and 300 nm was obtained (measured by ellipsometry, see SI Table S4). In the spotting method there are two possibilities: 1 µl drops were put onto a surface by handspotting. The concentration of the gel solution can be seen in Table 1. The total amount of gel solution was adapted to the required number of 1 µL spots. This method has the advantage of placing many spots on one substrate, which gives the option to study concentration variations or different biomolecules on one substrate. However, the gelification process is quite fast, which means that the time to place the drops is limited. The other possibility was to use a slide with 8 equally sized areas of 0.6 cm x 0.6 cm, confined by a Teflon template. They were filled with 10 µL solution each. This spotting gel solution was higher diluted (cf. Table 1), which slows down the gelification process. The hydrogel with PEG 6000 already showed to prevent leaching (see SI, Figure S3) and larger pores allow better diffusion of the target species. Therefore, it was used for the encapsulation of larger molecules such as streptavidin and the antibody in the following experiments. For the covalent encapsulation small oligonucleotide sequences of 20 bp were used and the probe covalently attached to the hydrogel. Because of that leaking is prevented inherently and


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diffusion of the complementary target sequence works for both hydrogels, consisting of PEG 3000 and PEG 6000. The swelling ratio α of these thin films was determined by measuring the layer thickness in dry and swollen state by atomic force microscopy (AFM). The hydrogel films are scratched to expose the surface of the substrate. The distance from the substrate to the hydrogel film was measured at three different points for each sample to obtain a mean value for ddry (cf. Table 2). The same holds for dwater, which is the thickness after incubating the substrate in water. From this data, the swelling ratio can be calculated applying equation (1). Table 2 shows the collected data, for each hydrogel two different thicknesses are investigated, the error was calculated as the propagation of uncertainty from the triplicate measurements. =

( )

(1)

( )

From Table 2 can be concluded, that G6 shows double the swelling ability than G3. The hydrogels are anchored covalently to the surface, which means that the swelling is reduced to only one dimension. Consequently, the height expansion is directly connected to the amount of water that is absorbed and corresponds to a larger overall pore volume. Table 2. Composition, layer thicknesses in dry state (ddry) and swollen in water (dwater), as well as the swelling ratio following equation 1.

Name

Components (molar ratio 3:1)

ddry

dwater

Swelling ratio α in water

G3

dPGN3 + PEG 3000

166

517

3.1 ± 0.5

G3

dPGN3 + PEG 3000

194

577

3.0 ± 0.1

G6

dPGN3 + PEG 6000

151

999

6.6 ± 1.4

G6

dPGN3 + PEG 6000

200

1139

5.7 ± 0.3

2.2 Non-covalent encapsulation Due to the chosen bioorthogonal strategy, it was possible to perform the gel formation process in the presence of a biomolecule. As a first model approach, streptavidin was chosen as probe molecule, and streptavidin-loaded hydrogel slides were prepared in the same way as the pure hydrogel by using a mixture of the gel components and streptavidin in water. Streptavidin was automatically encapsulated during the gel formation. The binding sites were not affected, and the streptavidin was homogeneously distributed and stably encapsulated in the hydrogel. This was shown by quantifying the amount of biotin which is diffusing into the hydrogel (cf. ACS Paragon Plus Environment


equation (2)) and plotting it against the amount of streptavidin that is immobilized within the hydrogel (c.f. Figure 2, right) for three different streptavidin concentrations as well as for a negative control where n (streptavidin) = 0 nmol. As a result, the slope reveals the number of accessible binding sites. In solution streptavidin offers 4 binding sites for biotin interaction, but it was shown that when immobilized on surfaces, only 2 of them are accessible.50 For the immobilization of streptavidin in our hydrogel we obtain a slope of 3.7 ± 0.5 (see SI for calculation and detailed description of the assay). The experiment was performed in triplicates and the error reflects the standard deviation. This means that our hydrogel matrix does not impede the streptavidin-biotin interaction and small molecules such as biotin can diffuse easily while the streptavidin stays inside the network. The negative control shows, that when there is no streptavidin, the biotin is easily washed out of the network. 1.80 1.60 1.40

n (biotinHydrogel) [nmol]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.20 1.00 0.80 y = 3.66x + 0.12 R² = 0.97

0.60 0.40 0.20

0.00 -0.10 0.00

0.10

0.20

0.30

0.40

0.50

n (streptavidin) [nmol]

Figure 2. Left: Scheme of the developed assay for the quantification of accessible binding sites. Streptavidin is captured inside the network, biotin is added, and excess biotin is washed out afterwards. Both amounts are quantified by quantitation kits. For detailed description see SI. Right: Plot of the amount of biotin against the amount of streptavidin encapsulated inside the hydrogel network. The slope of the line gives the number of accessible binding sites. The experiment was performed in triplicates, the error bars reflect the standard deviation.

For the use as a biosensor this streptavidin loaded hydrogel is spincoated onto a substrate and provides a ready-to-use streptavidin-hydrogel slide. Since biotin binds very strongly towards streptavidin, a solution of fluorescence-labeled biotin can be added and the fluorescence intensity of the hydrogel is analyzed. To evaluate the loading of the hydrogel slides with streptavidin, we compared the new system to a commercially available streptavidin slide, where the streptavidin was covalently coupled to an epoxy functionalized substrate. Both the commercial slide and the slide with the new ACS Paragon Plus Environment

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hydrogel embedded with streptavidin were processed equally with biotin solution, which was followed by the same washing procedure and the fluorescence determination. Figure 3 shows, that the fluorescence response of the hydrogel is drastically increased, contrary to a covalent attachment to the substrate. This is on one side attributed to the preservation of the binding sites inside the hydrogel network (shown in Figure 2), but the enhancement by a factor of 8 also validates the theory that we can enhance the signal intensity by a higher loading into the multiple layers of the 3D matrix.

Figure 3. Comparison of the determined fluorescence intensities of a commercial streptavidin slide and a slide with the encapsulated streptavidin in situ hydrogel film. The commercial slide (PolyAn GmbH) consists of a brush-like polymer bearing epoxy groups, where the streptavidin is immobilized by amine coupling chemistry.

In order to investigate the stability and to ensure that streptavidin was not leaching from the hydrogel we kept it in water for 2 weeks at room temperature and could show that the fluorescence did not drop significantly. Also, at higher temperatures up to 90 degrees, the hydrogel showed good performance (see SI, Figure S4). Besides streptavidin, the hydrogel should be suitable for any kind of biomolecule, due to its bioinert environment.51 To show the versatility of the hydrogel as a matrix for different biosensors we chose a system of antibody-antigen interaction, where the antibody (AntiFLAG-Antibody) is the encapsulated molecule and a fluorescence-labeled antigen (FLAG peptide) is the detection agent. Protein tags are peptide sequences genetically grafted onto a recombinant protein. Protein fusion tags like Strep-tag, HIS-tag or FLAG-tag are indispensable tools that are used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function.52-54 In recent years, numerous fusion tags have been developed. We chose for our experiments the FLAGTM fusion tag, which consists of eight amino acids including an enterokinase-cleavage site that was specifically designed for immunoaffinity chromatography.55 ACS Paragon Plus Environment


The slide preparation was performed by spotting the gel solution (cf. Table 1), with the antibody encapsulated. After adding the fluorescence-labeled FLAG peptide solution with an incubation time of 10 minutes followed by a washing procedure, the fluorescence intensity was determined with a fluorescence scanner. Figure 4 shows the increase in fluorescence intensity with increasing amount of immobilized ANTI-FLAG antibody. Furthermore, any unspecific binding could be excluded by applying FLAG peptide solution to a hydrogel slide without antibody, which showed no detectable signal.

Figure 4. Determination of fluorescence intensities for ANTI-FLAG antibody containing hydrogels with different amounts of antibody after treatment with FLAG peptide solution and a washing procedure. It can be clearly seen that the fluorescence intensity depends on the amount of encapsulated antibody, whereas the pure hydrogel shows no significant fluorescence (< 0.1 %). Error bars reflect standard deviation of three measured fluorescence intensities.

To investigate the sensitivity of the antibody slide towards the peptide, different concentrated peptide solutions were added to a slide with 2 µg antibody per square (0.6 x 0.6 cm2) and the fluorescence was measured. A very low peptide concentration of 2.5 nM can still be detected (cf. Figure 5).


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Figure 5. Determined fluorescence intensities after treatment of ANTI-FLAG antibody containing hydrogel slides with FLAG peptide solution of different concentrations. The inset shows a magnification for the lower concentrations to show that the very low concentration of 2.5 nM can be detected. The dashed line is the limit of detection which is determined as the mean value of the blank plus three standard deviations of the mean. Error bars reflect standard deviation of three measured fluorescence intensities.

2.3

Covalent binding

For systems, in which big molecules should be entrapped and small molecules should diffuse, the above-described non-covalent encapsulation method is a good choice. However, providing the possibility to entrap probes in a similar size as the target, the probe should be covalently anchored. For

this

purpose,

a

20mer

oligonucleotide

(probe)

functionalized

with

a

dibenzoazacyclooctyne (DBCO) unit can be clicked to the dPG-N3 hubs via SPAAC and be covalently bound into the hydrogel (Scheme 2). In principle, two pathways are possible: (1) the hydrogel is immobilized by spincoating to prepare a ready-to-use matrix on a substrate and the oligonucleotide is clicked into the network afterwards, or (2) the in situ approach is reapplied, in which case, all the single components are mixed to form the hydrogel in presence of the oligonucleotide.



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B

A

C

Scheme 2. To enable the fast and efficient reaction with the hubs, the oligonucleotides are functionalized with a dibenzoazacyclooctyne (DBCO) unit and reacted together with the PEG during gel formation (A). Azidefunctionalized 2D and 3D slides were prepared to compare the new hydrogel system with the spincoated hydrogel substrates. See SI for further information (B). Schematic presentation of the in situ preparation of a hydrogel, in which the probe oligonucleotides are covalently linked to the hubs of the gel network. Afterwards, a fluorescence-labeled target oligonucleotide can be detected (C).

The loading behavior was studied and the performance of the spincoated hydrogel matrix was compared to the performance of 2D (monolayer of azide groups) and 3D slides (brush like polymers) as shown in Scheme 2. In all cases, the same DBCO bearing oligonucleotide was immobilized following the same protocol. As expected, the 2D slides with only a monolayer of azide groups available for reacting with the DBCO-modified oligonucleotides showed the poorest performance. The 2D slides showed the lowest fluorescence signal throughout the whole concentration range (cf. Figure 6).


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Gel

2D-slide

3D-slide

rel. fluorescence (target oligonucelotide)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

10

20

30

40

50

probe oligonucleotide conc. [µM] Figure 6. Relative fluorescence intensities to compare the performances of the 2D, 3D slides and the hydrogel considering probe concentrations ranging from 0.05-51 µM. Error bars reflect the standard deviation of three measured fluorescence intensities. Lines are for the guidance of eye.

However, both the 3D slide and the hydrogel slide successfully used the third dimension to enhance the signal intensity. The performances of both are comparable for oligonucleotide concentrations below 10 µM. For higher concentrations, the 3D slide system is restricted by the spatial limitations imposed by the brush-like structure to further immobilize hybridization accessible probe strands. This does not hold for the hydrogel, which combines a large number of functional groups per surface area with a solution like environment. As a result, the hydrogel-based system can incorporate more probe strands that are well accessible for hybridization. Figure 7 shows that the fluorescence signal increases with prolonged diffusion time but reaches saturation after approximately 4 h, which could in some cases be unfavorable. To save preparation time and further increase the fluorescence response, the in situ method was applied. Here the immobilization of the probe strand is conducted in solution, where there is no diffusion limitation and the SPAAC reaction between DBCO oligonucleotide and the hubs of the hydrogel is very fast. This reduces the reaction time from 4 h to several minutes and ensures a better distribution throughout the layers of the network.


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rel. Fluorescence (target oligonucleotide)


0.5

1

2

4

16

probe oligonucleotide immobilization time [h]

Figure 7. Effects of prolonged immobilization time of the probe oligonucleotide in a constant concentration for all time intervals of 12 µM into the preformed hydrogel and subsequent hybridization overnight.

The even higher fluorescence response for the in situ method is shown in Figure 8, where the same concentration of probe strand is immobilized for the different immobilization methods and substrates.

Figure 8. Comparison of the determined fluorescence intensities of oligonucleotides immobilized in a concentration of 50 µM on a 2D, 3D, and two hydrogel slides. The “hydrogel slide functionalized after gelation” shows the result when spotting the oligonucleotide into the already formed hydrogel, while “hydrogel slide in situ method” on the right reports the fluorescence intensity when mixing the components beforehand and the oligonucleotide is clicked into the gel before its formation.


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Since the same amount of biomaterial gives a more pronounced signal, the efficacy of such a biosensor is greatly improved. This is due to the immobilization in solution and the distribution throughout all the layer of the gel network, which cannot be guaranteed for in the post gelation immobilization method. The in situ prepared gel is not only more effective for higher but also for lower concentrations of the probe strand. In a sensitivity study (see SI, Figure S5) a constant excess target concentration of 500 nM was hybridized with probe molecules immobilized in 1 µL spots with concentrations ranging from 0.05 µM to 1.6 µM. We show that probe concentrations of 0.05 µM still give sufficiently high fluorescence intensities for a significant detection in case of the in situ hydrogel. The signals of the 2D and 3D slides, however, were too low to be considered as a positive signal. We think this is due to the solution like environment of the gel. This ensures that every probe can be reached by the target. Also, the efficiency of the click reaction during the immobilization process seems to be poorer when it occurs in close proximity to the surface in case of the 2D and 3D slides. As a result less probe material is needed for the production of in situ hydrogel sensor chips To determine the sensitivity towards the target, oligonucleotide strands were spotted on the 2D and 3D slides as well as into the spincoated gel at the same concentration of 13 µM for all spots and subsequently hybridized with target concentrations of 250 nM, 125 nM, 62.5 nM, and 31.25 nM (see Figure 9). Again, the loaded hydrogel showed by far the best performance and could still detect oligonucleotide solutions of only nanomolar concentrations. The high sensitivity towards the target oligonucleotide allows one to circumvent the costly procedure of doing the enrichment beforehand. An example for integral cost savings would be the application of the new materials in Point of Care Devices (PoC). Typically, such a device consists of several parts or partial areas: i) the sample preparation step (enrichment, purification, filtration), ii) the part of the (bio)molecular interaction, i.e. the detection of the analyte and iii) the part of the readout or signal generation and processing. In case of using the surface bound hydrogels, the first part i) would be omitted, which would reduce the technical complexity as well as the complexity of the device, reduce the (technical) footprint and thus enable further miniaturization and probably also shorten the analysis time.


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rel. fluorescence intensity


250

125

63

31

targetconc. [nM] Gel

3D-N3

2D-N3

Figure 9. Determined fluorescence intensities after treatment of oligonucleotide-containing (13 µM) hydrogel, 2D and 3D slides with the target oligonucleotide solution of different concentrations. Error bars are standard deviations of 4 spots.

To investigate the stability of the oligonucleotide-loaded hydrogel, the idea was to denature the hybridized strands and prove by rehybridization that the target strand can diffuse in and out of the gel, whereas the probe strand remains covalently attached to the gel. Therefore, it was exposed to either heat or a strong base (NaOH), followed by a washing procedure and drying under a stream of compressed air. By repeating this process five times, whereby every hybridization was done for 2 h at the stated concentration, the gel showed the fluorescence evolution depicted in Figure 10.


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B)

Gel

rel. fluorescence

A)

rel. fluorescence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3D Slide 2D Slide

hyb.

denat.

Figure 10. (A) Five cycles of hybridization in the given concentration (hyb), denaturation utilizing the given method (denat) and rehybridization (rehyb) for one gel with previously mixed oligonucleotide to follow the ability of regeneration and stability during heating processes and exposure to sodium hydroxide (NaOH). (B) The comparison with 2D and 3D slides shows that a denaturation process of 85 °C for 15 minutes works for the gel but is not sufficient for 2D and 3D slides.

Surprisingly, the second hybridization, which is the first rehybridization, shows a higher fluorescence signal than before, although the concentration and reaction time remained equal. This effect was also observed in other experiments not only for the gel but also for the linkerbased 2D and 3D slides, which is also described in the literature.56 Most probably, it is attributed to the previous treatment with heat, which results in a reorganization and improved orientation of the oligonucleotides. Additionally, the present polymer network structure is not a rigid structure. The mesh size is influenced by the swelling of the polymer chains. There are changes in the PEG structure ranging from fully elongated to slightly accumulated depending on the amount of water and the temperature. The elongation of the PEG chains alters the pore sizes inside the hydrogel and that influences the availability of the probe strands. The treatment with sodium hydroxide did not lead to an increase in fluorescence intensity, which reinforces the hypothesis that the enhancement is connected to the swelling of the gel. The overall enhancement in fluorescence intensity is not considerable enough to always treat the slides with heat, since the expense of an additional preparation step is not worth the reward. Comparing the degeneration of the hybridized strands in the hydrogel with the 2D and 3D slides shows that the degeneration for the 2D and 3D slides was not as effective as for the new 3D hydrogel, for which only 15 minutes of exposure to 85 °C was enough to almost extinguish the signal for the target oligonucleotide strand. This is attributed to the solutionACS Paragon Plus Environment


like environment created by the hydrogel, which facilitates the efficiency of the denaturation process whereby the proximity to the surface of the 2D and 3D slides apparently impedes the process and some of the fluorescence labeled target molecules remain stuck on the surface.

3

Conclusion

Within this work, we successfully investigated a new biosensor matrix for in situ hydrogel formation with simultaneous encapsulation of biomolecules without any unspecific interaction between the gel components and the biomolecule. For big molecules like streptavidin and the ANTI-FLAG antibody, we chose the non-covalent encapsulation, whereas, in the case of a 20mer oligonucleotide, it was covalently anchored to the hubs of the gel network. In both cases, we achieved a much higher loading as well as better sensitivity when the hydrogel is compared to commercial 2D and 3D slides. The in situ hydrogel shows long-term stability even under harsh conditions like heating or treatment with base. Furthermore, no bleeding of the encapsulated biomolecules was observed while the 4 binding sites of streptavidin are preserved. The bioorthogonality of the hydrogel as well as the solution like environment kept the biomolecule stable in its native state and ensures a good accessibility for the affinity interaction by smaller, in the case of streptavidin and ANTI-FLAG antibody, or similar sized molecules, in the case of the oligonucleotides. Spotting the hydrogel is greatly advantageous because only a very small amount of material is needed. A professional spotter makes it possible to have many hydrogel spots with different entrapped biomolecules on one surface. The principle strategy of the in situ hydrogel immobilization concept is not limited to the already analyzed examples, but can be expanded to different applications. This provides a great opportunity to create cheap, stable, and highly sensitive biosensors for diverse applications.


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4

Acknowledgement

The authors are grateful to Dr. Jose Luis Cuellar Camacho for operating the AFM.

5

Supporting Information

•

Chemical synthesis and analytics of the synthesized components

•

Surface treatment and gel preparation

•

Analytics of the gels (contact angle, AFM, ellipsometry)

•

Encapsulated streptavidin (preparation of streptavidin slides, treatment with biotin solution and washing procedure, loading with streptavidin, stability study)

•

Encapsulated ANTI-FLAG antibody (preparation of antibody slides, treatment with peptide solution and washing procedure)

•

Covalently

immobilized

oligonucleotides

(oligonucleotide

characterization, sensitivity study)


processing

and


6

References

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