Responsive Photonic Crystal Carbohydrate Hydrogel Sensor

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Responsive Photonic Crystal Carbohydrate Hydrogel Sensor Materials for Selective and Sensitive Lectin Protein Detection Zhongyu Cai, Aniruddha Sasmal, Xinyu Liu, and Sanford A. Asher ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00426 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 23, 2017

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Responsive Photonic Crystal Carbohydrate Hydrogel Sensor Materials for Selective and Sensitive Lectin Protein Detection tection Zhongyu Cai, Aniruddha Sasmal, Xinyu Liu*, Sanford A. Asher* Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA

*Corresponding author. E-mail: [email protected]; [email protected] KEYWORDS Photonic crystals; Carbohydrate hydrogels; Biosensors; Lectin proteins detection; Copolymerization.

ABSTRACT: Lectin proteins, such as the highly toxic lectin protein, ricin, and the immunochemically important lectin, jacalin, play significant roles in many biological functions. It is highly desirable to develop a simple but efficient method to selectively detect lectin proteins. Here we report the development of carbohydrate containing responsive hydrogel sensing materials for the selective detection of lectin proteins. The copolymerization of a vinyl linked carbohydrate monomer with acrylamide and acrylic acid forms a carbohydrate hydrogel that shows specific “multivalent” binding to lectin proteins. The resulting carbohydrate hydrogels are attached to 2-D photonic crystals (PCs) that brightly diffract visible light. This diffraction provides an optical readout that sensitively monitors the hydrogel volume. We utilize lactose, galactose and mannose containing hydrogels to fabricate a series of 2-D PC sensors that show strong selective binding to the lectin proteins ricin, jacalin and concanavalin A (Con A). This binding causes a carbohydrate hydrogel shrinkage which significantly shifts the diffraction wavelength. The resulting 2-D PC sensors can selectively detect the lectin proteins ricin, jacalin and Con A. These unoptimized 2-D PC hydrogel sensors show a limit of detection (LoD) of 7.5 × 10-8 M for ricin, a LoD of 2.3 × 10-7 M for jacalin, and a LoD of 3.8 × 10-8 M for Con A, respectively. This sensor fabrication approach may enable numerous sensors for the selective detection of numerous lectin proteins.

Proteins and carbohydrates are two of the central building blocks of life. Protein-carbohydrate interactions play fundamental roles in many biological processes, such as cell recognition and cell adhesion, and in the etiology of a broad range of diseases.1-4 The binding between individual proteins and carbohydrates is generally nonspecific and relatively weak, with association constants in the range of µM to mM.5 Fortunately, the specificity and affinity of protein-carbohydrate interaction can be dramatically enhanced by the clustering of multiple carbohydrates, a phenomenon defined as the “cluster glycoside effect”.6, 7 Multivalent protein-carbohydrate interactions are widely utilized in nature to achieve strong and specific molecular and cellular interactions.8 For example, many highly poisonous toxins, including Shiga-like toxin and ricin, induce cellular toxicity through multivalent interactions.9, 10 Numerous approaches that use multivalent binding have been developed to detect and to monitor proteincarbohydrate interactions both in vitro and in vivo.4, 11, 12 Water-soluble polymer,13 dendrimers14-16 as well as solid carriers11, 17 such as nanoparticles, 11, 18-22 carbon nanotubes17 and graphene23 were used as scaffolds to present multivalent carbohydrate ligands. These systems were utilized as biosensor devices and bioimaging agents for lectins,23-26 toxins,19-22, 27 microbes and tumor cells.28-32 These systems are quite versatile;33-36 they were applied to

sense and quantify protein-carbohydrate interactions by additionally utilizing fluorescence, NMR, circular dichroism, surface plasmon resonance spectroscopy, total internal reflection fluorescence spectroscopy and microscopy.12, 33 Unfortunately, the need for these sophisticated instrumentation approaches to detect protein-carbohydrate interactions restricts their utility for use in limited resource environments. The development of a simple and inexpensive method to monitor protein-carbohydrate interactions is highly desirable. Recently, there has been significant interest in the use of photonic crystals (PCs) for sensing due to their intrinsic simplicity and their diffraction efficiency.37-47 3-D PC sensors were developed to sense many species, including metal ions, glucose, sarin and various saccharides. 38-42, 45 A major limitation of nonclose packed 3-D PC hydrogel sensors is that the electrostatic self-assembly of highly ordered arrays requires very low ionic strength conditions, which can be inconsistent with the sensor fabrication chemistry.44 To surmount this difficulty, we developed a 2D PC sensing technology that avoids the need for low ionic strength conditions.43, 44 This 2-D PC sensing motif uses a monolayer array that is prepared independently of the responsive hydrogel. We monitor the shift in diffraction of the 2-D array attached to the surface of the responsive hydrogel. This approach is simple and highly efficient. We

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used this approach to develop sensors for pH, surfactants, metals, proteins and microbes.43, 48-55 In this study, we synthesized carbohydrate containing hydrogels for developing sensors for lectin proteins, such as the highly toxic lectin protein, ricin, and the immunochemically important lectin, jacalin.10, 56 These sensor materials utilize our 2-D PC sensing motif to quantify the concentrations of lectin proteins. As discussed in detail below we first self-assemble a 2-D PC at the air-water interface and transfer it onto a glass slide. We then polymerize a vinyl linked carbohydrate monomer solution containing acrylamide and acrylic acid onto the 2-D PC monolayer. This forms the sensing responsive hydrogel sensor material. These responsive hydrogels contain carbohydrates attached to a polymer backbone that multivalently binds lectin proteins. This causes a hydrogel shrinkage that blue shifts the diffraction wavelength of the 2-D PC array attached to the hydrogel surface. The vivid optical diffraction reports on the lectin protein concentration.

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thesized. The synthesis protocol is shown in the supporting information (Synthesis protocols are shown in Supporting Information, Schemes S4 and S5). The 1H and 13C NMR spectra of these two carbohydrate monomers and their analysis are shown in Figures S1-S5. The structure of commercial allyl α-D-galactose and allyl β-D-galactose was also confirmed with 1H NMR spectra (Figures S6 and S7).

Experimental Section Materials and Methods. Allyl α-D-galactose and allyl β-D-galactose were purchased from Sigma-Aldrich and Fisher Scientific, respectively, and used as received. The seeds of the castor oil plant can produce two similar proteins, ricin (RCA60) and an agglutinin (RCA120). The less toxic RCA120 were used in this study. Unconjugated ricinus communis agglutinin I (RCA I, RCA120) and unconjugated jacalin were purchased from Vector Laboratories, Inc., and were prepared at the required solution concentrations prior to use. Acrylamide (AAm), acrylic acid (AAc), N,N’methylenebisacrylamide (MBAAm), 2-hydroxy-1-(4-(2hydroxyethoxy)-phenyl)-2-methyl-1-propanone (Irgacure 2959), dimethyl sulfoxide (DMSO), 2-allyloxyethanol, styrene, HEPES, sodium azide, sodium chloride, calcium chloride dehydrate, bovine serum albumin and manganese chloride tetrahydrate were purchased from Sigma-Aldrich, and used as received. D-lactose monohydrate and D-mannose were purchased from Chem-Impex International Inc. Hg(CN)2, HgBr2, and BF3•Et2O were purchased from Acros Organics. Deuterium oxide was purchased from Cambridge Isotope Laboratories, Inc. Concanavalin A (Con A) was donated by Sigma-Aldrich. 1-Propanol was purchased from J. T. Baker Inc. Monodisperse, ∼650 ± 16 nm diameter polystyrene (PS) particles were synthesized by using an emulsifier free emulsion polymerization as previously reported.57 Fabrication of 2-D PC Lectin Protein Sensors. 2Allylethoxyl-β-D-lactose was synthesized from commercially available D-lactose in 3 steps (Synthesis protocols are shown in Supporting Information, Schemes S1-S3). Mannose monomer (2-allylethoxyl α-D-mannose) with the same 2-allylethoxy linker at its reduced ends was also syn-

Figure 1. Illustration of the fabrication of 2-D PCCarbohydrate hydrogel sensors.

Figure 1 shows the procedures for the fabrication of our 2-D PC PAAm-AAc-Carbohydrate hydrogels. The 2-D PS colloidal PC array was first assembled on a water surface by using our needle tip flow technique.48 The 2-D PC array on the water surface was then transferred to a glass slide and dried in the air. A 50 µL solution of AAm, AAc, MBAAm, and 2-allyloxyethyl lactose (or other carbohydrate monomer) was layered onto the 2-D array on the glass slide (24 × 50 mm2). Another glass slide was placed on top to cover and flatten the polymerization solution. The copolymerization was carried out by using 365 nm UV light (UVP, UVGL-55 handheld UV Lamp, 6-Watt) at room temperature. After 20 min, we peeled the 2-D array hydrogel film from the glass slide and washed it at least 5 times in 10 mM phosphate buffered saline (PBS) solution (containing 0.15 M NaCl) to remove any unreacted monomers and impurities. Scheme 1 illustrates the copolymerization of PAAm-AAc-Carbohydrate hydrogels. A series of 2D PC lactose hydrogels were fabricated by using monomer solutions containing 20, 40 and 80 mg/mL of 2-allylethoxyl lactose. These samples are denoted as 2-D PC PAAm-AAcLactose-20, PAAm-AAc-Lactose-40, PAAm-AAc-Lactose-80, respectively. Likewise, the galactose and mannosecontaining hydrogels are similarly denoted. The different reaction stoichiometries utilized for the 2-D PC carbohydrate hydrogel preparations are listed in Table 1.


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Scheme 1. Synthesis of PAAm-AAc-Carbohydrate hydrogels (R represents vinyl or allyl substituted carbohydrates).

Table 1. Stoichiometry of 2-D PC PAAm-AAc-Carbohydrate hydrogelsa

Samples

AAm (mg)

Carbohydrate monomer (mg)b

MBAAm (mg)

AAc (µL)

2-D PC PAAm-AAccarbohydrate-20

40

8

0.4

8

12


40

16

0.4

8

12


40

32

0.4

8

12


40

48

0.4

8

12


40

64

0.4

8

12

Irgacure 2959 (µL) c

a The total reaction solution volume is 400 µL; b The carbohydrate monomers used in this study include 2-allylethoxyl-β-Dlactose, allyl α-D-galactose, allyl β-D-galactose and 2-allylethoxyl α-D-mannopyranoside; c The initiator is Irgacure 2959 in DMSO (33 %, w:v).

Protein Recognition. The response of these sensors to proteins was characterized by using either a UV-Vis reflection spectrometer, or by light diffracted from a green laser pointer (λ=532 nm). For the PAAm-AAc-Lactose hydrogel, the hydrogel sensors were first equilibrated in 10 mM PBS solution (containing 0.15 M NaCl and 0.08 wt% sodium azide) at pH 7.8 for 24 h, during which the PBS solution was frequently changed. Then small pieces of the sensor (8 mm × 8 mm squares) were placed in 0 to 1.0 mg/mL ricin solutions containing 0.15 M NaCl and 0.08 wt% sodium azide. The 2-D PC PAAm-AAc-Lactose sensors were equilibrated overnight before diffraction measurements. Bragg diffraction from the 2-D PCs on the PAAm-AAcLactose hydrogel sensors was monitored by using an Ocean Optics USB2000-UV-VIS Spectrometer, a LS-1 Tungsten Halogen Light Source and an R-series Fiber Optic Reflection Probe. All diffraction measurements were carried out with the 2-D PC-Carbohydrate hydrogels on a silver front surface mirror (Thorlabs, VA). The diffraction measurements were carried out in a Littrow configuration with the fiber at a ~14o angle from the array normal.43 The response of other carbohydrate hydrogels was measured in the same way. For the detection of Con A with the PAAm-AAc-Mannose hydrogel, the hydrogel sensors were pre-equilibrated with

a 0.1 M NaCl solution containing 1 mM Ca2+ and Mn2+. The Con A sample solutions were prepared with a 0.1 M NaCl solution containing 1 mM Ca2+ and Mn2+. For the detection of jacalin using the PAAm-AAc-Galactose hydrogel, the hydrogel sensors were equilibrated with 10 mM HEPES buffered saline at pH 8.5 containing 0.1 mM Ca2+ and 0.08 wt% sodium azide. For the reversibility study, the 2-D PC PAAm-AAcLactose hydrogel sensors were immersed into a 2 mL 1.0 mg/mL ricin solutions containing 10 mM PBS at pH 7.8 for 4 h before each measurement. After the Debye diffraction ring diameter measurements, the samples were washed with a large amount of 10 mM PBS at pH 7.8 prior to the next round of measurements. Microscopy and NMR Spectroscopy Characterization. The carbohydrate monomers were characterized with 1H NMR and 13C NMR (Bruker Avance III 600MHz and 400MHz), respectively. The 2-D PS colloidal PC arrays and the 2-D PC PAAm-AAc-Carbohydrate hydrogel sensors were sputter-coated with gold (Au). The surface morphology measurements were taken by using a scanning electron microscope (SEM, JEOL JSM6390LV).

Optical Diffraction Characterization. Under the irradiation of a laser pointer, the 2-D PC diffracts light at an angle that depends on both the interparticle spacing


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and the laser wavelength.58-60 The rotationally disordered small 2-D PC array domains (20 × 20 μm2) diffract a Debye ring pattern. The first-order diffraction angle, α, depends upon the particle spacing: sin α = 2λlaser/(31/2d), where α is the interior angle of the Debye diffraction ring. λlaser is the laser wavelength, and d is the particle spacing. The diffraction angle α, is determined from the Debye ring diameter: α = tan−1(D/2h), where D is the Debye ring diameter and h is the distance between the 2-D PC array and screen. We monitor the 2-D PC array particle spacing by measuring D and h and calculating : d =

4λlaser ( D / 2) 2 + h 2

.49 In this

3D study, the h value was set to 36 mm. For each analyte concentration, 3 identical samples were used and each sample was measured at 3 different positions. The average and standard deviation of d were obtained from these 9 values. The standard deviation of the particle spacing is calculated using the following equation:

1 1

The diffraction of our 2-D PC PAAm-AAcCarbohydrate hydrogel sensors was also measured by using an Ocean Optics USB 2000-UV-Vis spectrometer, a LS-1 tungsten halogen light source, and an R-series fiber optic reflection probe in a Littrow configuration with the fiber probe at a ∼14° angle from the array normal. The Debye diffraction ring diameter measurement physically involves the same diffraction process as that measured by the UVVis reflection probe fiber optic spectrometer. In a Littrow configuration, the 2-D Bragg diffraction relationship is mλ = 31/2d sin θ, where m is the diffraction order, λ is the diffracted wavelength (in vacuum), d is the 2-D particle spacing, and θ is the angle of the incident light relative to the 2D PC array normal.60, 61

Results and Discussion

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Figure 2. (a) SEM image of 2-D PCs of monodisperse PS particles with a diameter of ∼650 nm; (b) Photograph of diffraction from 2-D PCs on the surface of a PAAm-AAc-Lactose hydrogel (~80 µm thick) illuminated with a flashlight below at an angle of ∼50° to the normal. The white light forward diffraction gives rise to a spectrum of colors; (c) SEM image of 2-D PCs on a freshly prepared 2-D PC PAAm-AAc-Lactose hydrogel attached to a cover glass and dried in air prior to Au sputtering; (d) SEM image of 2-D PCs on a swollen 2-D PC PAAm-AAcLactose hydrogel that was attached to a cover glass and dried in air before Au sputtering. The PAAm-AAc-Lactose hydrogel adhered to the cover slide, which prevented its area from shrinking. This prevented contraction of the expanded hydrogel 2-D array upon drying.

Figure 2a shows an SEM image of a 2-D PC fabricated on an air-water interface, which was transferred onto a glass slide, and then dried in air. The resulting 2-D PC is highly ordered over dimensions of 20 µm × 20 µm. Cracks in the hexagonal ordering presumably occur as the 2-D array shrinks as it dries on the slide. Figure 2b shows the white light diffraction spectrum of a 2-D PC PAAm-AAc-Lactose hydrogel when illuminated from below by a collimated white light source incident at 50° from the normal. The diffraction colors vary from the deep red to blue. This diffraction spectrum is similar to that of the 2-D PC on the glass slide shown in Figure S8a. Figure 2c shows an SEM image of an almost close packed 2-D PC on the surface of an ~80 µm thick PAAm-AAcLactose hydrogel sensor that was dried in air. The 2-D PC remains well ordered and almost close-packed (Figure 2c). This hydrogel sensor was removed from the glass slide, swollen in a 0.1 M NaCl solution, and then dried onto a glass slide. During drying the swollen hydrogel surface adheres to the glass slide which prevents the hydrogel area from shrinking. As a result, the hydrogel can only shrink along its thickness. Thus, the 2-D array spacing is larger in Figure 2d compared to Figure 2c. The analyte lectin protein induced volume response of the carbohydrate hydrogel sensor suspended in water can be determined, by measuring its white light diffraction shifts. We can monitor the 2-D PC diffraction wavelength maximum by using a reflection optical fiber probe in the Littrow configuration, as we previously demonstrated.43 Alternatively, we can irradiate the 2-D PC carbohydrate hydrogel sensor along its normal with a green laser pointer. The light is forward diffracted into a Debye ring as shown in Figure S8b. The Debye diffraction ring diameter is directly proportional to the 2-D array nearest neighbor spacing.49, 51, 55 Both diffraction methods can be used to monitor changes in the hydrogel volume that give rise to changes in the 2-D array spacing.51, 53, 55 Generally, Debye ring diffraction measurements are more convenient. Figure 3a shows the dependence of the diffraction spectrum of the 2-D PC PAAm-AAc-Lactose-80 hydrogel sensor on different concentrations of ricin. These diffraction spectra were measured with an Ocean Optics reflection fiber probe in a Littrow configuration. The diffraction wavelength maximum of the 2-D PC PAAm-AAc-Lactose-80 sensor shifts from 622 nm to 583 nm as the ricin concentration increases from 0 to 1.0 mg/mL. The inset photographs


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show the colors of the forward-diffracted light taken with a camera along the normal and with a white light source below at an angle of ~76° to the 2D array normal. The diffraction color changes from red to green as the hydrogel sensor is exposed to increasing ricin concentrations from 0 to 1.0 mg/mL. Figure 3b shows the ricin concentration dependence of the particle spacing of the 2-D PC PAAm-AAc-Lactose hydrogel sensors measured by using the Debye diffraction rings. These Debye ring particle spacing measurements are consistent with those measured by using the reflection fiber probe (Figure 3a). The 2-D particle spacing decreases as the ricin concentration increases because multivalent ricin binding to the hydrogel lactose increases the hydrogel crosslink density. This shrinks the hydrogel causing a 2-D array particle spacing decrease. This array spacing decrease blue shifts the 2-D PC Bragg diffraction. Figure 3b also shows that larger particle spacing decreases occur as the hydrogel lactose concentration increases. A 16 nm particle spacing decrease occurs upon exposure of the 2-D PC PAAm-AAc-Lactose-20 hydrogel sensor to 1.0 mg/mL ricin, while a larger 51 nm particle spacing decrease occurs upon exposure of the 2-D PC PAAm-AAc-Lactose-40 hydrogel sensor to 1.0 mg/mL ricin. The 2-D PC PAAm-AAc-Lactose120 hydrogel sensor shows the largest particle spacing decrease (~124 nm) for a ricin concentration of 1.0 mg/mL. The increasing diffraction blue shifts due to hydrogel lactose concentration increases occur because an increased lactose concentration increases the binding between lactose and ricin, which gives rise to more crosslinks, and thus, a larger hydrogel shrinkage. The increased binding results from both an increased lactose concentration and an increased affinity caused by multivalent binding. Thus, a higher lactose content leads to a larger responsivity of the 2-D PC PAAm-AAc-Lactose sensor. We determined a limit of detection (LoD) of the 2-D PC PAAm-AAcLactose-120 hydrogel sensor of 9 µg/mL (7.5 × 10-8 M) ricin, which is far below the adult human lethal oral dose of 20-30 mg/kg.62 This LoD value is comparable to a mouse bioassay method (∼7.5 µg/mL) but our ricin sensor responds faster (100 min) compared to the mouse bioassay method (24 h).63, 64 We also determined that the dynamic range of the 2-D PC PAAm-AAc-Lactose-120 hydrogel sensor is between 2.5 × 10-7 M to 24.3 × 10-7 M, while its linear range is from 7.5 × 10-8 M to 27.5 × 10-8 M (see SI and Figure S9 for calculation details).

Figure 3. (a) Normalized and smoothed diffraction spectra of 2-D PC PAAm-AAc-Lactose hydrogel sensors for different ricin concentrations. These measurements were taken in a Littrow configuration with an angle of 14° between the probe and the normal to the 2-D array. The Littrow configuration occurs when the diffracted light is detected in back diffraction. In the Littrow configuration, the 2-D Bragg diffraction relationship is mλ = 31/2d sin θ, where m is the diffraction order, λ is the diffracted wavelength (in vacuum), d is the 2-D particle spacing, and θ is the angle of the light relative to the normal to the 2-D array.60 (b) Ricin concentration dependence of 2-D PC PAAmAAc-Lactose hydrogel particle spacing changes.

Figure 4 shows the lectin protein and the BSA concentration dependence of the particle spacing of the 2-D PC PAAm-AAc-Lactose-40 hydrogel sensor measured by using the Debye diffraction ring diameter. The 2-D array particle spacing decreases with increasing ricin concentrations. This response is due to selective ricin binding to lactose as evident from the control experiments, where little change occurs upon addition of the proteins BSA, or the other lectin proteins jacalin and Con A. Proteins that do not bind to lactose do not give rise to any diffraction shifts. Thus, this 2-D PC PAAm-AAc-Lactose sensor selectively and sensitively detects ricin.65, 66


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Figure 5. Ricin concentration dependence of 2-D PC PAAmAAc-β-Galactose-40, PAAm-AAc-α-Galactose-40, and PAAmAAc-Lactose-40 hydrogel sensor particle spacing changes.

Figure 4. Dependence of 2-D PC PAAm-AAc-Lactose-40 hydrogel particle spacing on BSA and the lectin proteins ricin, Con A and jacalin. Only ricin binding blue shifts the diffraction.

We further fabricated 2-D PC PAAm-AAc-Galactose and 2-D PC PAAm-AAc-Mannose hydrogels using similar synthetic methods (Scheme 1). Figure 5 shows the ricin concentration dependence of the particle spacing of 2-D PC PAAm-AAc-β-Galactose-40, PAAm-AAc-α-Galactose-40, and PAAm-AAc-Lactose-40 hydrogel sensors. At 1.0 mg/mL ricin concentrations, the α-galactose, β-galactose and lactose hydrogel sensors show 2, 15 and 51 nm particle spacing decreases, respectively. As expected, the PAAm-AAc-α-Galactose-40 hydrogel sensor shows the smallest response because ricin has a negligible binding affinity to α-galactose. In contrast, ricin (RCA60) is known to specifically bind to β-galactose with a single binding site that has an association constant of ∼6900 M-1. Ricin (RCA60) has larger affinity for lactose since ricin has two binding sites for lactose with association constants of Ka1=35000 M-1 and Ka2=2800 M-1.67 The RCA120 used in this study has two binding sites for βgalactose and four binding sites for lactose, respectively, since it consists of two RCA60, in which the two RCA60 (A-B) chains are covalently connected by a disulfide bond.67, 68 The multiple hydrogel lactose monomers enhance multivalent binding between lactose and the RCA120 ricin protein, which enables sensitive detection of lectin ricin.

In this work, we also examined the selectivity of our mannose and α-Galactose hydrogel sensors towards other lectin proteins, such as Con A that binds mannose and jacalin that binds α-galactose.52, 69, 70 Figure 6a shows the Con A concentration dependence of the particle spacing change for 2-D PC PAAm-AAc-Mannose and 2-D PC PAAm-AAc-Lactose hydrogels. The 2-D PC PAAm-AAc-Mannose hydrogel shows a much larger response to 2.0 mg/mL Con A (∼124 nm particle spacing decrease) due to the strong Con A binding to mannose. Only a ∼4 nm particle spacing decrease occurs for the 2D PC PAAm-AAc-Lactose hydrogel. We fabricated more sensitive Con A sensors by simply increasing the mannose concentration to 160 mg/mL (see Figure S10 in SI). The calculated Con A detection limit of this unoptimized 2-D PC PAAm-AAc-Mannose-160 hydrogel sensor is 3.8 × 10-8 M. The dynamic range of the 2-D PC PAAmAAc-Mannose-160 hydrogel sensor is between 1.3 × 10-7 M to 29.1 × 10-7 M, while its linear range is from 3.8 × 10-8 M to 42.3 × 10-8 M (see SI and Figure S10 for calculation details). Our unoptimized 2-D PC PAAm-AAc-Mannose160 hydrogel sensor shows higher sensitivity and a larger linear range than the reported colorimetric method for Con A detection, which was reported to have a LoD of 10-7 M and a linear range from 8 × 10-8 M to 26 × 10-8 M (Table S1 in SI). 71 We synthesized the α-galactose carbohydrate hydrogel for sensing jacalin. Figure 6b shows the highly selective response of the 2-D PC PAAm-AAc-α-Galactose sensor to the lectin jacalin. A ∼65 nm particle spacing decrease occurs when the PAAm-AAc-α-Galactose hydrogel sensor is exposed to 2 mL of a 1.0 mg/mL solution of jacalin. In contrast, a negligible ∼2 nm particle spacing decrease is observed for the 2-D PC PAAm-AAcLactose hydrogel sensor to jacalin. This is because of jacalin’s much larger association constant to αgalactose (2.2 ± 0.8 × 107 M-1) compared to lactose and mannose. 70, 72 By increasing the α-galactose concentration to 160 mg/mL (see Figure S11 in SI), we fabricated a more sensitive jacalin sensor. We determined a detection limit of 2.3 × 10-7 M for this unoptimized 2-D PC PAAm-AAc-α-Galactose-160 hydrogel sensor towards jacalin. The dynamic range of the 2-D PC PAAm-AAc-αGalactose-160 hydrogel sensor is between 7.6 × 10-7 M to 55.8 × 10-7 M, while its linear range is from 2.3 × 10-7 M to 3.8 × 10-7 M. To the best of our knowledge, this is the first sensor for jacalin detection (see SI and Figure S11 for calculation details).


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ACS Sensors The kinetics of 2-D PC PAAm-AAc-Lactose hydrogel sensor response to 1.0 mg/mL ricin is shown in Figure 8. The PAAm-AAc-Lactose hydrogel sensor particle spacing decrease saturates ~100 min after exposure to 2 mL of a 1.0 mg/mL ricin solution. This response follows a single exponential decay e-kt (k ∼0.033 min-1). This relatively slow response is presumably limited by the slow diffusion rate of ricin (RCA120, Mw=120,000 Da) into the hydrogel which limits the rate of its multivalent crosslink formation.

Figure 8. Kinetics of 2-D PC PAAm-AAc-Lactose-40 hydrogel to ricin at a concentration of 1.0 mg/mL. The hydrogel is 80 µm thick.

Figure 6. (a) 2-D PC PAAm-AAc-Mannose-40 and 2-D PC PAAm-AAc-Lactose-40 hydrogel sensors for the selective detection of Con A, and (b) 2-D PC PAAm-AAc-Lactose-40 and 2D PC PAAm-AAc-α-Galactose-40 sensors for the selective detection of jacalin.

We probed the reversibility of our 2-D PC carbohydrate hydrogel sensors. The response of our PAAm-AAc-Lactose hydrogel sensor over 5 cycles of exposure to 0 and 1.0 mg/mL ricin solutions is highly reversible (Figure 7). The particle spacings are relatively constant before and after ricin addition.

Conclusions We report the development of carbohydrate hydrogel sensor motif for the sensitive and selective detection of lectin proteins. A 2-D PC array was attached to a hydrogel containing lectin protein specific carbohydrate to fabricate the sensor motif. Selective multivalent lectin protein binding to the hydrogel carbohydrate forms crosslinks, which shrink the hydrogel volume. This blue shifts the diffraction of the 2-D PC sensor. The 2-D PC PAAm-AAc-Lactose sensor selectively detects ricin and shows a ricin detection limit of 9 µg/mL (LoD=7.5 × 10-8 M). The α-galactose hydrogel sensor is selective for jacalin (LoD=2.3 × 10-7 M), while the mannose hydrogel sensor is highly selective for Con A (LoD=3.8 × 10-8 M). These sensor motifs show very bright diffraction readouts, which may enable visual biological and chemical agent detection in applications such as food safety, healthcare, and chemical threat monitoring.

Acknowledgements We thank Dr. Zhenmin Hong for helpful discussion, Mr. Kyeongwoo Jang for 1H NMR measurements and SigmaAldrich for their generosity in providing expensive chemicals. The authors gratefully acknowledge HDTRA (Grant No. 1-151-0038 to S. A.) and University of Pittsburgh (to X. L.) for financial support.

ASSOCIATED CONTENT Figure 7. Reversibility of the 2-D PC PAAm-AAc-Lactose-40 hydrogel for ricin sensing.

Synthesis protocols and 1H NMR and 13C NMR of carbohydrate monomers, photographs of 2-D photonic crystals and Debye diffraction ring, and details on the calculations of limit of detection, dynamic range and linear range of the photonic crys-


ACS Sensors

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tal hydrogel sensors are included into Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

*Emails: [email protected]; [email protected].

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Responsive Photonic Crystal Carbohydrate Hydrogel Sensor

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