Differential Stability of Biosensing Proteins on Transferred Mono

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Differential Stability of Biosensing Proteins on Transferred Mono-/bilayer Graphene Bo Hou, and Adarsh Radadia ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00379 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 27, 2018

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ACS Biomaterials Science & Engineering

Differential Stability of Biosensing Proteins on Transferred Mono-/bilayer Graphene Bo Hou, Adarsh D. Radadia Institute for Micromanufacturing, Center for Biomedical Engineering and Rehabilitation Services, Louisiana Tech University, 911 Hergot Ave, Ruston, Louisiana 71270, United States Corresponding Author * Adarsh D. Radadia, E-mail: [email protected]. Tel.: +001 3182575112.

KEYWORDS Enzyme stability, graphene bio-functionalization, graphene surface chemistry, glucose oxidase, horseradish peroxidase

ABSTRACT

Graphene, due to its outstanding electrical and optical properties, has been attractive to develop biosensors and bioelectronics. The stability of proteins on graphene, as a function of its secondary structure, has been studied computationally; however there has been a lack of experimental validity of such simulations results. This study examines the stability of two

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biosensing enzymes on graphene and in solution: horseradish peroxidase (an all α-helix protein), and glucose oxidase (a protein with both α-helix and β-sheet content). At three different temperatures (4, 20, 37 °C) glucose oxidase tethered to graphene was found to be more stable than when in solution. In contrast, horseradish peroxidase tethered to graphene showed rapid loss in activity than when in solution. This is the first experimental evidence showing differential stability of proteins on graphene and we believe this is due to the difference in the secondary structure of the proteins.

Introduction Graphene based electrical1-7 and optical8-11 biosensors have been heavily investigated over the last decade, incorporating selective protein elements such as enzymes, and antibodies. Electrical detection of DNA down to 1 pM12, and E. coli as low as 10 cfu/mL13, along with real-time detection of glucose and glutamate molecules at clinically relevant concentrations has been demonstrated using graphene based biosensors14. More recently, an experimental study using a goat anti-human IgG and a simulation study using a Mus musculus IgG suggested graphene is an ideal candidate for IgG attachment because the IgG attaches to graphene in an end-on configuration where the Fc region attaches to graphene and both Fab regions sticks out for antigen-binding15. However, the stability of the biosensing proteins, which affects the sensor life expectancy with graphene is an important, and often over-looked limiting factor. Such data is needed to validate the development of nanoelectronic biosensors with graphene films, especially graphene produced via CVD because it allows the production of mono-/bilayer graphene for nanoelectronic biosensors with better continuity and reproducibility.


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The interaction of proteins with graphene has been studied extensively via simulations but rare experimentally. As deposited CVD graphene is an uncharged hydrophobic surface, which according to molecular dynamics simulations could force ordering of water layers (hydrophobic hydration) that results in hydrogen bond fluctuations, density oscillations, and dipole biasing of water molecules16-19. Intuitive non-covalent interactions between proteins and graphene such as van der Waals and π- π stacking20-21, along with hydrophobic hydration-mediated interactions make protein stability on graphene a complex problem that needs to be investigated experimentally and theoretically22-25. Simulations accounting for hydrophobic hydration suggest a tight water layer trapped between graphene and β-sheet structure, which prevents the attraction of the protein to graphene26. The α-helices are lesser stiff secondary structures compared to the β-sheets; hence the protein stability on graphene can be predicted based on the α-helix and βsheet content in the secondary structure of the protein. Additionally, the conformational changes observed for a protein adsorbed on graphene have been found to be a strong function of the protein orientation during adsorption24. Despite much of the simulation effort, there is a lack of systematic experimental effort comparing the stability of proteins on graphene to that in solution. The few experimental studies of protein degradation on graphene that have been reported so far show that the degradation of both types of proteins, all α-helix and all β-sheet proteins on graphene. FTIR studies show that the α-helical structure of an adsorbed peptide on dry graphene surface distorts in the presence of water27. In contrast, experimental work by Ditchel group on an antibody as well as Concanavalin A, both of which are all-β sheet structures have been shown to lose selectivity on graphene; the primary reason was believed to be denaturation of the proteins on graphene28-29.


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Here, we report the stability of two model enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP) on mono-/bilayer graphene films and compare it when the enzymes are in solution. Enzymes were chosen for this protein stability studies because enzyme activity is sensitive to minor changes in its secondary structure and the technology to measure activity of these enzymes is matured and commercially available. GOx and HRP were chosen for this study because they are among the most commonly used biosensing enzymes whose stability in solution and other substrates has been reported. Present literature lacks the data on stability of GOx and HRP on graphene. HRP is a 44 kDa, heme-containing, all α-helical glycoprotein extracted from horseradish. Purified HRP is relatively unstable compared to when conjugated to proteins such as immunoglobulins. On the contrary, GOx from Apergillus Niger is a 160 kDa, dimeric, hemecontaining, glycoprotein containing both α-helices and β-sheets. In addition to the structural property of the enzyme, the stability of surface-tethered enzymes also depends on the chemical nature of the surface and the bio-immobilization chemistry. This choice for nanoelectronic biosensors, especially biomolecular field-effect transistors (BioFETs) has been limited to non-covalent chemistries involving polycyclic aromatic hydrocarbons in order to avoid substantially tailoring the electronic or optical property of graphene. 1pyrenebutanoic acid, succinimidyl ester (PBASE) is one such compound, which has frequently been used to prepare carbon nanotube-based BioFETs for the last two decades, and since inception it has been used in preparing graphene-based BioFETs routinely30-31. The use of PBASE linker allows π- π stacking between the pyrene rings and the graphene surface, and the attachment of the succinimidyl ester group to the NH2- residues on proteins. Many pyrene-based compounds have been synthesized for use with graphene. An example includes work by Ditchel group where pyrene-based “multi-pod” compounds (compounds with multiple pyrene rings


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allowing multiple anchors to graphene) were synthesized for biofunctionalization of graphene2829

. Other non-covalent biofunctionalization routes include the use of protein conjugated metal

nanoparticles, biotin-streptavidin pair, and multi-functional polymers32-34. Regardless, PBASEassisted biofunctionalization continues to be the primary method of preparing graphene nanoelectronic biosensors. In this paper, we use the PBASE-assisted tethering of GOx or HRP to graphene and commercial activity measurement assays to gauge its stability relative to freshly functionalized form. Material and Methods Materials Mono-/bilayer graphene grown by low-pressure chemical vapor deposition on copper foil was purchased from Graphene Labs. Poly(methyl methacrylate) (996 kDa) and anisole (≥99%) were obtained from Sigma-Aldrich. Copper etchant (CE-100) was obtained from Transene, Inc. The methanol, 2-propanol, 30% H2O2 and 29% NH4OH were obtained CMOS grade from J.T. Baker. The 39% HCl was obtained from Fisher Scientific. The 99.8% dichloromethane (HPLC grade) stabilized with methanol, and dimethyl sulfoxide (DMSO) were obtained from ACROS Organics. HRP (303 U/mg) was from Thermo Scientific, and its activity was measured using a proprietary TMB formulation (Sure Blue Reserve™) from Kirkegard & Perry Labs. As received HRP was diluted to 0.1 mg/mL with 0.1 M potassium phosphate buffer (pH 6.0) prior to aliquot and storage at -20 °C. GOx and a fluorometric GOx assay kit were obtained from Abcam (ab138884). As received GOx was also diluted to 100 U/mL with 0.1 M potassium phosphate buffer (pH 6.0) prior to aliquot and storage at -20 °C. PBASE was obtained from Setareh Biotech.


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Graphene Transfer The method published by Liang et al. was adapted to transfer graphene from copper to 5 × 5 mm square silicon pieces with a 285-nm thick thermal oxide layer35. A 5% PMMA solution in anisole was spin coated on one side of the copper foil at 3000 rpm for 30 seconds, and baked at 110 °C for 2 min. The graphene on the backside of copper was etched completely using oxygen plasma (100 W, 260 mTorr, 20 sccm, 30 s). The exposed copper foil was etched using copper etchant at 40 °C for 1 h. The resulting graphene-PMMA composite was cleaned with a modified SC-2 process (20:1:1::H2O:30%H2O2:39%HCl, 15 min at 20 °C). The cleaned graphene-PMMA was then transferred to the 5 × 5 mm Si/SiO2 substrates, which had been cleaned with the standard

SC-1

(5:1:1::H2O:30%H2O2:29%NH4OH,

75

°C,

10

min),

and

SC-2

(5:1:1::H2O:30%H2O2:39%HCl, 75 °C, 10 min). The resulting graphene films was annealed in 2.5%H2/97.5%Ar at 400 °C for 1 h. Supporting Information Figure S1 shows the optical and scanning electron microscopy of sample graphene films used in this study. Enzyme Functionalization on Graphene A fresh 5 mM PBASE solution was prepared (28.9 mg 1-pyrenebutanoic acid, succinimidyl ester in 15 ml di-methyl sulfoxide) to dip substrates with graphene for 1 h at 20 °C in a dark chamber. Excess PBASE was removed by soaking in DI water on a microplate shaker at 80 rpm for 5 min twice. Chips were then dried using a gentle stream of nitrogen. A 10 µL aliquot of enzyme solution was spotted to cover the entire 5 × 5 mm substrate surface. A coverslip was used on the top to minimize evaporation as well as to make sure the enzyme solution only touched the graphene coated surface. After 1 h incubation at 20 °C, chips were soaked in PBST20 solution (pH 7.4) on a microplate shaker at 80 rpm for 5 min twice and in potassium


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phosphate buffer for 5 min twice. Thus, prepared chips were stored in potassium phosphate buffer until tested. The activity of HRP and GOx were analyzed using reaction schemes shown in Figure 1. All experiments in this paper were carried out with potassium phosphate buffer (0.1 M, pH 6.0, 20 °C) due to its low variation in pH with temperature (d(pKa)/dT = -0.0028).

Figure 1. Overall scheme of reactions used to detect activity of HRP and GOx on CVD graphene. Testing stability of HRP in solution The HRP activity was measured using a colorimetric assay involving the oxidation of a highly sensitive commercial formulation of 3,3’,5,5’-tetramethylbenzidine, called SureBlue Reserve™ (SRB), for 20 min at 20 °C, followed by its quenching with 1 M HCl and measurement of optical density (OD) at 450 nm. In each case the OD of 100 µL SRB was used to subtract background prior to adding 50 µL of HRP solution of appropriate concentration. A unit of HRP was defined as amount required to form 1 mg of purpurogallin from pyrogallol in 20 s at 20 °C and pH 6.0. First, the calibration curve relating the OD and various amounts of HRP (5, 10, 15, 20, 25, 50, 75, 100 pg) were obtained through three repeats. Secondly, the concentration dependent stability


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of HRP was measured in triplicates using 25, 50, 75, and 100 pg HRP in 50 µL for times extending up to 24 h. Thirdly, the temperature dependent stability of HRP was measured using 100 pg of HRP in 50 µL at 4 °C, 20 °C, and 37 °C for times up to 12 h.

Testing stability of HRP on graphene Four HRP concentrations were selected to plot out the calibration curve and find the linear range of enzyme amount on graphene. First, 100 µL of SureBlue Reserve™ was added in wells and optical density was measured as background. Afterward, chips were inserted in wells carefully so that the SureBlue Reserve™ substrate can fully wet the chips. Reaction happened at 20 °C for 20 minutes. Then, chips were removed and optical density was measured again. The concentration of 0.3 mg/ml was chosen to observe the activity performance of HRP within 12 h at 3 temperatures (4 °C, 20 °C, and 37 °C). For tests on graphene there were two ways to setup the experiment: (1) Test the activity of enzymes on the same chip at different time points: This approach overcomes the variability in enzyme loading per chip. However, we found that the enzyme activity assay conditions negatively impact the stability of enzymes and hence this method could not be used to measure enzyme stability on graphene. (2) Simultaneously prepare identical chips functionalized with enzymes, store them at same conditions, and draw three chips to measure the enzymatic activity at different time points. The chips were discarded after measuring the enzyme activity. Thus, the method (2) was used and the variability in enzyme amount per chip was addressed by carrying out the experiments with three repeats and averaging the data for each time point. Testing stability of glucose oxidase in solution


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One unit of GOx was characterized as amount required to oxidize 1 µmole of β-D-glucose to D-gluconolactone per min at pH 6.0 at 30 °C. Activity of GOx was quantified using a commercial GOx activity assay kit where the fluorescent resorufin (Figure S2) was detected at Ex/Em = 540/590 nm with a fluorescence micro-plate reader. In the experiment of measuring glucose oxidase amount in solution, the RFU values for glucose oxidase concentrations between 0.1 and 10 mU/ml fell in the linear range. The higher concentration (10 mU/ml) was chosen for studying the deactivation of glucose oxidase at 20 °C at 0, 1, 2, 3, 4, and 10 days. In order to get high fluorescence readings with low errors, experiment of glucose oxidase stability at different temperatures (4 °C, 20 °C, and 37 °C) was carried out at concentration of 10 mU/ml. Firstly, RFU of reaction mixture was measured as background, and then glucose oxidase solution at various concentrations was added to the reaction mixture. Reaction happened in the microplate reader with continuous shaking at 37 °C. After 15-minute reaction, microplate reader read the fluorescence signal another time. The final relative fluorescence units were calculated by deducting the background information from the second fluorescence reading. Testing stability of glucose oxidase on graphene Similar to the experiment of testing the stability of HRP on graphene, observing the stability of glucose oxidase on graphene was achieved via the same enzyme treatment process. At the beginning, 6 concentrations of glucose oxidase (0.001, 0.01, 0.1, 1, 10, 100 U/ml) were selected to plot out the calibration curve and find the linear range of concentrations on graphene by adding 50 µL of reaction mixture in wells. Afterward, chips with graphene on top were inserted in wells with facing down so that the reaction mixture can fully wet chips. Reaction happened at 37 °C in 15 minutes. Then, chips are removed and RFU was measured the second time. The con-


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centration of 10 U/ml was chosen to observe the activity performance of glucose oxidase within 10 days at 3 temperatures (4 °C, 20 °C, and 37 °C). Data was collected at 0, 1, 2, 3, and 10 days.

Results and Discussion HRP activity assay Figure 2a shows that the OD values read from the HRP activity assay were close to a linear function of HRP content in solution. This allowed us to directly use OD as means to interpret amount of HRP that was active. Next, in Figure 2b the activity of HRP monitored for times extending up to 24 h in solution allowed us to gauge how long to conduct HRP stability studies on graphene, and check for concentration dependence of HRP stability in solution. As shown in Figure 2b, regardless of the HRP solution concentrations, more than 50% of HRP activity was lost in 9 h, following an integrated rate law for first order reaction, ln(CA0/CA) = kt with a rate constant k of 0.087 ± 0.005 h-1. After 24 h, nearly 80% of the HRP activity was lost in solution. Thus, further HRP stability study on graphene and in solution were restricted to 12 h. Next, HRP activity on graphene was measured, and the OD reading was used to quantify amount of HRP tethered to graphene (Figure 2c) using the calibration data in Figure 2a. The amount of HRP attached to graphene was linearly dependent on the HRP amount applied indicating a functionalization efficiency of 0.006%. No plateau or saturation loading of HRP was detected. This implies that crowding related suppression of HRP activity can be safely neglected.


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Thus, further experiments were carried out by applying 3 µg of HRP on graphene substrates, which was expected to attach about 70 HRP molecules per µm2.

Figure 2. (a) HRP activity measured as OD at 450 nm found to be linear with respect to the weight of HRP in a 50 µL aliquot at 20 °C. (b) HRP activity measured as a function of time at 20 °C in a 50 µL aliquot with either 25, 50, 75, or 100 pg of HRP. (c) Amount of HRP tethered to graphene measured as a function of HRP amount applied during the functionalization procedure. Comparison of HRP activity in solution and on graphene when stored at (d) 4 °C, (e) 20 °C, and (f) 37 °C in 0.1 M potassium phosphate buffer (pH 6.0, 20 °C) for times up to 12 h. Solution samples consisted of 100 pg HRP in a 50 µL aliquot. Graphene samples were functionalized with 3 µg HRP. Relative OD is calculated as the background-corrected OD for the stored sample divided by that recorded for a freshly functionalized sample. Results were obtained by averaging data from three repeats. The error bars indicate the standard deviation.


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HRP stability in solution and on graphene Figure 2d-f show the activity of HRP measured when in solution and on graphene at 4 °C, 20 °C, and 37 °C for times up to 12 h. As expected, HRP activity in solution and on graphene is observed to diminish rapidly at increasing temperature, with most stability seen at 4 °C. Our results in Figure 2d-f show that activity of HRP drops faster on graphene than when in solution at 4 °C and 20 °C, while at 37 °C the drop in HRP activity was found to be similar. The activity loss due to desorption of HRP from graphene is neglected due to the stringent washing routine adopted in our studies and we attribute the drop in activity to the denaturation of HRP. The deactivation of enzymes can be considered a reaction of nth order with reaction rate constant k. This allows us to write the general differential rate law as −

=

(1),

where CActive Enzymes is the concentration of active enzymes. Since the OD is linearly proportional to the concentration of the active enzymes, we can rewrite (1) as −

!

= "#

(2).

Thus, plotting ln (-dOD/dt) versus ln (OD) allows calculating the order and the rate constant of the reaction from the slope and the intercept, respectively. However, like most enzyme


12

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deactivation, we found that HRP degradation followed a first order kinetics and thus eq. (2) took the form of $% &

!'

!'(

) = − *

where a plot of relative OD (

(3), !'

!'(

) on semi-log plot versus t would be linear with a slope of (-

k).

Figure 3. Semi-log plot of relative OD versus time for OD data obtained from HRP stability tests in solution (a) and on graphene (b) at 4 °C (black), 20 °C (blue), and 37 °C (red). The error bars indicate the standard deviation from three repeats. The dashed lines show a fit to eq. (3); refer to Table 1 for k-values. The HRP degradation in solution as well as on graphene was found to follow a first order reaction kinetics as seen in Figure 3. Assume an Arrhenius equation applies, ln (k) = - Ea / RT + ln (A), where k is rate constant, A is the pre-exponential factor, Ea is the activation energy, R is the universal gas constant (1.987 cal/mol-K), and T is absolute temperature (K). Table 1 lists the values for Ea and A that were calculated. As can be noticed, the lower value for A on graphene indicates the low frequency of denaturing interactions; this can be attributed to the lower degrees


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of freedom to move for an immobilized enzyme on graphene. The higher error for Ea on graphene arises from the higher standard deviation within the data. Regardless, the HRP deactivation process faces a lower activation barrier on graphene as compared to when in solution. Our results for HRP, which is an all α-helical protein are in full agreement with those obtained experimentally by Katoch et al.27, and theoretically by Guo et al. for α-helix structures on graphene24. Table 1. The rate constant (k) calculated when data in Figure 3 fit to first order kinetics. 4 °C (h-1)

20 °C (h-1)

37 °C (h-1)

Ea

A (h-1)

(kcal/molK) 0.035 0.009

± 0.089 0.006

± 0.188 0.027

±

9.9 ± 1.1

2.3 × 105

On Graphene 0.063 0.007

± 0.148 0.005

± 0.197 0.025

±

5.7 ± 1.7

2.1 × 103

In Solution

GOx activity assay GOx was quantified in terms of its activity (Unit) as defined earlier rather than its weight. Supporting Information Figure S2 shows that the fluorescence readings from the GOx activity assay were close to a linear function of GOx concentration between 0.1 and 10 mU/ml. Further, the stability of GOx in solution has been known to be independent of its concentration36-37. Like prior studies38-39, the GOx stability tests in our case were also conducted for a 10-day period. The GOx activity on graphene was measured as a function of GOx amount incubated on graphene during the functionalization step. As shown in Supporting Information Figure S3, the


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GOx activity obtained on graphene showed a plateau or saturation loading of GOx past 100 mU. To avoid crowding-related suppression of GOx activity, functionalization of graphene for stability studies was carried out by applying 100 mU GOx, which falls on the linear portion of curve in Figure S3.

GOx stability in solution and graphene surface

Figure 4. Comparison of GOx activity in solution and on graphene when stored at (a) 4 °C, (b) 20 °C, and (c) 37 °C in 0.1 M potassium phosphate buffer (pH 6.0, 20 °C) for times up to 10 days. (d), (e), and (f) show statistical comparison of GOx activity in solution and on graphene at


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different temperatures for days 2, 3 and 10, respectively. Blue bars indicate GOx activity measured on graphene. Red bars indicate GOx activity measured in solution. The p-values are reported from a paired t-test (two-tailed). Solution samples consisted of 100 pg HRP in a 50 µl aliquot. Graphene samples were functionalized with 100 mU GOx. Relative fluorescence was calculated as the background corrected fluorescence units recorded for the stored sample divided by that recorded for a freshly functionalized sample. Results in this figure were obtained by averaging data from three independent experiments. The error bars indicate the standard deviation.

Figure 4 compares the average activity of GOx measured when on graphene to that recorded in solution at 4 °C, 20 °C, and 37 °C for times up to 10 days. Statistically the GOx activity was similar in solution and on graphene for days 2 and 3; however statistically significant (p

Differential Stability of Biosensing Proteins on Transferred Mono

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