Deflection cantilever detection of interferon gamma

Accepted Manuscript Title: Deflection cantilever detection of interferon gamma Author: Remko van den Hurk Stephane Evoy PII: DOI: Reference:

S0925-4005(12)00935-5 doi:10.1016/j.snb.2012.09.023 SNB 14545

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

21-3-2012 4-9-2012 9-9-2012

Please cite this article as: R. van den Hurk, S. Evoy, Deflection cantilever detection of interferon gamma, Sensors and Actuators B: Chemical (2010), doi:10.1016/j.snb.2012.09.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Deflection cantilever detection of interferon gamma Remko van den Hurk* and Stephane Evoy

Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta,

ip t

a

Canada, T6G 2V4

cr

*Corresponding Author

us

*Contact Information: Remko van den Hurk

an

Email Address: [email protected] Phone Number: (780) 641-1681

M

Fax Number: (780) 492-1811

Abstract:

d

Interferon-gamma (IFN-γ) is a biomarker protein and an indicator of relapse in multiple

te

sclerosis (MS) patients. Treatment of diseases and conditions like MS may be time sensitive, and deflection cantilevers are a promising platform for real-time measurement of biomarker proteins.

Ac ce p

Additionally, linking procedures have a substantial effect on the sensitivity of deflection cantilever measurements. Therefore, successful detection of IFN-γ using three different linking procedures is presented. Enzyme-linked immunosorbent assays were used to confirm the efficacy of the linking procedures before they were implemented on cantilever deflection arrays. The steady state change in surface stress due to IFN-γ binding was determined to be 0.16±0.09, 0.11±0.04 and -0.08±0.06 N/m for the glutaraldehyde, Prolinker B and 1-Ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide (SulfoNHS) linking procedures, respectively. Furthermore, R2 values near 1 indicate good correlation between an exponential model for antibody-antigen binding and cantilever deflection. Steady state deflection of 667.3±0.1, -452.0±0.0, and -406.2±0.2 nm and rate constants of 0.4582±0.0003, 0.4737±0.0000 and 0.1766±0.0002 1/hours were calculated for the glutaraldehyde, Prolinker B and EDC/Sulfo-NHS linking procedures, respectively.

Page 1 of 26

Keywords: Cantilever, deflection, protein detection, sensor, linking, interferon gamma

1 Introduction Biomarkers can be used to determine the presence of various diseases, disease severity,

ip t

degree of progression of chronic conditions and effectiveness of treatments. Examples of protein biomarkers include Tau protein for Alzheimer's disease, cardiac reactive protein (CRP) for heart

cr

disease, prostate specific antigen (PSA) for prostate cancer and interferon gamma (IFN-γ) for multiple sclerosis (MS)[1-4]. These biomarkers, and in fact the majority of biomarkers, are not

us

individually specific to a disease or condition however. It is often necessary to determine the levels of multiple biomarkers in order to provide accurate diagnoses. The cytokine IFN-γ, for

an

example, is a biomarker for MS, but is also a biomarker for other diseases such as tuberculosis and lupus erythematosus[5, 6]. In fact, panels of cytokines are of interest as biomarkers for numerous diseases and conditions including the studies on tuberculosis and lupus erythematosus.

M

Surface acoustic wave, surface plasmon resonance, quartz crystal microbalance and cantilever biosensors are of interest for the detection and measurement of biomarkers.

d

Advantages of biosensors over more common immunoassays include label-free measurement and ease of multiplexing. Cantilevers are of particular interest because they are easy to fabricate,

te

can perform rapid real-time measurements, can be fabricated into large arrays to simultaneously

Ac ce p

measure multiple analytes, and can be miniaturized for incorporation into lab on a chip applications[7].

Deflection cantilevers are well suited to the detection of biological elements because they operate well in aqueous solutions. As the sample solution flows across the cantilever, the biomarker binds specifically to the functionalized surface. This induces a differential surface stress, which causes the cantilever to deflect. The deflection can then be related to the biomarker concentration through various transduction methods. Aside from biomarkers, there are a variety of other possible applications of deflection cantilevers sensors. A number of these applications are in the growing field of food and water safety[8]. Both operation in solution and large arrays would be advantageous for toxin detection in water treatment plants, for example. Practical application would require significant future work however[8].

Page 2 of 26

A variety of different proteins have been investigated using deflection cantilevers. Specific detection of PSA[9-11], CRP[10, 12], vimentin[13], glutathione s-transferase[14], myoglobin[15, 16], creatin kinase[15], a human estrogen receptor[17], cyclin dependent protein kinase 2[18], taq polymerase[19], human immunodeficiency virus type 1 glycoprotein 120 (gp

ip t

120)[20], a peptide[21], streptavidin[22], different antibodies[23-25], and human interleukin1β[26] has been demonstrated.

cr

In the majority of the experiments, antibodies were used to bind the protein of interest[916, 20, 21, 24-26]. Other specific capture methods include using an antigen to detect an

protein of interest[19], or biotin to detect streptavidin[22].

us

antibody[23], peptides specific to the protein of interest[17, 18], a DNA sequence specific to the

an

Various methods were used to bind the capture molecules to the cantilevers. Linkers with succinimide leaving groups were the most frequently reported. These included dithiobis (sulfosuccinimidylpropionate) (DTSSP), di-thio-bis-succinimidyl undecanoate (DSU), N-

M

hydroxysuccinimide (NHS), Biotin-SS-NHS and Biotin-SS-Sulfo-NHS. DTSSP and DSU form monolayers of free succinimidyl groups on gold surfaces[9, 11, 15, 25]. NHS was used in

d

conjunction with a monolayer of carboxyl groups and 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), a crosslinker which improves the efficacy of NHS[20, 23, 24]. Biotin-SS-

te

Sulfo-NHS was used to link biotinylated antibodies to the surface through neutravidin[11].

Ac ce p

Biotin-SS-NHS was used to capture streptavidin[22]. Thiolation of the capture molecule is another common linking method for gold surfaces [14, 16-19, 21]. Less common linking methods include using a calixcrown[10], polyethyleneimine[13], glutaraldehyde on a carboxylated surface[26], and biotin linkers (N-(6(Biotinamido)hexyl)-3’-(2’-pyridyldithio)-propionamide (Biotin-HPDP) and biotin-polyethylene glycol disulfide (Biotin-PEG) to detect streptavidin[22]. According to Shu et al., the linking molecule has a substantial affect on the magnitude and direction of the cantilever deflection due to biotin-streptavidin binding[22]. Similarly, Lam et al. observed a significant decrease in differential deflection due to gp 120 binding when a PEG spacer group was inserted between the capture antibody and the cantilever surface [20]. In this report we present, to our knowledge, the first cantilever-based detection of IFN-γ. More specifically, multiple linking procedures were investigated in order to assess their impact on cantilever deflection measurements. The effectiveness of the linking procedures was initially

Page 3 of 26

examined using an enzyme linked immunosorbent assay (ELISA). Following the ELISA experiments, the linking procedures were implemented on cantilever arrays. Cantilever deflection was used to calculate the surface stress induced by IFN-γ binding, and a theoretical equation was fit to the difference in deflection between the active and reference cantilevers. The

ip t

ELISA and microcantilever platforms agree that the Prolinker B-and glutaraldehyde-based

linking chemistries offer superior reliability compared to the EDC/Sulfo-NHS-based linking

cr

chemistry.

us

2 Materials and Methods

2.1 Reagents & Solutions

an

The human IFN-γ ELISA kit from R & D Systems contained rabbit anti-human IFN-γ capture antibody (hC-Ab), human IFN-γ standard, goat anti-human IFN-γ detection antibody (D-

M

Ab), and streptavidin horseradish-peroxidase (streptavidin-HRP). Stop solution (2 N H2SO4), color reagent A (H2O2), color reagent B (Tetramethylbenzidine) and rabbit anti-canine IFN-γ

d

capture antibody (cC-Ab) were also obtained from R&D Systems. The 96-well plates were obtained from BD Falcon. The 95% isopropyl alcohol (IPA) and 99% ethanol were acquired

te

from University of Alberta chemistry stores. Water, aside from that used during gold etching and

Ac ce p

piranha cleaning, was filtered using the MilliQ water purification system from Millipore. Water, gold etch, H2SO4, H2O2 and IPA used during the gold etch and piranha cleaning steps were obtained from the University of Alberta Nanofab. The 2-[methoxy(polyethyleneoxy) propyl]trimethoxysilane (PEG-Silane) was obtained from Gelest. Prolinker B was acquired from Proteogen. EDC and Sulfo-NHS were obtained from Fisher Scientific. The remaining chemicals and reagents were obtained from Sigma-Aldrich. The piranha solution consisted of a 3:1 v/v mixture of 86% H2SO4 and 30% H2O2. Phosphate buffered saline (PBS) solution consisted of 0.2 μm-filtered 2.7 mM KCl, 8.1 mM Na2HPO4, 137 mM NaCl, 1.5 mM KH2PO4, pH 7.3. PB and low NaCl concentration PBS were identical to the PBS solution but with 0 mM and 20 mM NaCl respectively. Concentrated PBS consisted of 0.2 μm-filtered 1 M NaCl, 270 mM KCl, 810 mM Na2HPO4, and 150 mM KH2PO4, pH 7.3. The 2-(N-morpholino)ethanesulfonic acid saline (MES) buffer contained 0.1 M 2-(Nmorpholino)ethanesulfonic acid and 0.5 M NaCl, pH 6.0. Low concentration MES buffer

Page 4 of 26

contained 10 mM 2-(N-morpholino)ethanesulfonic acid and 38 mM NaCl, pH 5.5. Reagent diluent contained 0.2 μm-filtered 0.1% bovine serum albumin, 0.05% Tween 20, 20 mM Trizma base, 150 mM NaCl, pH 7.3 in water. The substrate solution was a 1:1 mixture of Color reagent

ip t

A and Color reagent B. Wash buffer consisted of 0.05% Tween 20 in PBS, pH 7.3.

2.2 Equipment

cr

Double side polished silicon wafers were obtained from University Wafer. Silicon and gold-coated silicon cantilever arrays were obtained from IBM. The arrays each held 8 cantilevers

us

measuring 500±3 μm x 100±3 μm x 1.0±0.1 μm with a Young’s modulus of 169 GPa±5%[27]. The gold-coated silicon arrays arrived pre-evaporated with 3 nm chromium and 20 nm of gold. The Cantisens platform from Concentris was used to measure the deflection of the cantilevers in

an

the array. A functionalization unit from Concentris with 150 μm diameter glass capillaries was

M

used to functionalize the cantilevers.

2.3 ELISA Procedure

d

ELISAs were performed to determine the effectiveness of the linking procedures. The experiments were performed in 96-well plates with silicon chips with 3 nm of titanium and 40

te

nm of gold evaporated on each side. The chips were sonicated in acetone, washed in IPA and

Ac ce p

water, sonicated in 99% chloroform, and washed in chloroform. The Prolinker B chips were placed in 1 mM Prolinker B for 1 hour, washed in chloroform, IPA, water and 3x in PBS, and placed in 4 μg/ml hC-Ab for 1 hour.

The glutaraldehyde chips were washed in water and 3x in PBS, placed in 10 mM cysteamine in PBS overnight, and washed 3x in PBS. Subsequently, the chips were incubated in 10% v/v glutaraldehyde in PBS for 1 hour, washed 3x in PBS and incubated for 1.5 hours in 4 μg/ml hC-Ab in PBS.

The EDC/Sulfo-NHS chips were washed 3x in water and placed in 10 mM cysteamine in water overnight. They were then washed in water and 3x in PBS. Subsequently, a solution with 2 mM EDC, 5 mM Sulfo-NHS and 4 μg/ml hC-Ab in MES buffer was prepared. After 15 minutes, 20 mM 2-mercaptoethanol was used to inactivate the EDC, and the pH was raised above 7.0 through the addition of 20% by volume concentrated PBS. The solution was used to functionalize the aminated gold surface for 1 hour.

Page 5 of 26

The remainder of the ELISA procedure was identical for each linking procedure. The IFN-γ, D-Ab, HRP-Streptavidin and substrate solution steps were preceded by a 3x rinse in wash buffer. The chips were incubated in: 1000, 250, 62.5, or 0 pg/ml IFN-γ in reagent diluent for 2 hours, followed by 2 hours in 50 ng/ml D-Ab in reagent diluent and 20 minutes in 1:200 v/v

ip t

HRP-Streptavidin:reagent diluent. Following the wash, the chips were moved to new wells. This was necessary because proteins bind to both the chips and the surface of the wells. The original

cr

wells and the new wells were then filled with 200 μl Substrate Solution. After 20 minutes 100 μl of Stop Solution was added to each well, the chips were removed from the new wells and the

us

optical density at 450 nm was recorded with an ELISA plate reader.

an

2.4 Cantilever Functionalization

For the glutaraldehyde linking procedure, pre-evaporated gold was removed by washing the array in gold etch and water, dipping it in a solution of 1.125 M NaOH and 1.5% v/v H2O2 in

M

water and washing it again in water. The array was dried with nitrogen, cleaned in piranha solution for 20 minutes, washed in water and IPA, and dried with nitrogen. The top surface of the

d

array was evaporated with 5 nm titanium and 40 nm of gold, and was incubated in 10 mM cysteamine-HCl in PBS overnight. The array was washed 3x in PB, submerged in 2% PEG-

te

silane in ethanol for 40 minutes, and washed 3x in ethanol and 2x in water. It was then placed in 2% glutaraldehyde in PB at pH 7.5 for 2 hours and washed 3x in water. The whole array was

Ac ce p

incubated in 100 μg/ml C-Ab in PB for 2 hours, and washed 3x in PB. The even cantilevers were blocked with 10 μg/ml IFN-γ for 1.5 hours and washed 3x in PB. For the Prolinker B linking procedure, the silicon array was cleaned in piranha solution for 20 minutes, washed in water and IPA, and dried with nitrogen. Hydrolyzation of 2% by volume PEG-Silane in ethanol was allowed to proceed for 5 minutes. The chips were placed in the PEG-Silane solution for 2 minutes, dipped in ethanol and baked at 110°C for 15 minutes. After cooling, the chips were washed in ethanol. A 3 nm titanium adhesion layer and a 40 nm layer of gold were evaporated onto the active surface of the cantilever array. The array was then incubated in 3 mM Prolinker B in chloroform for 1 hour, was washed in chloroform, acetone, IPA, water and ethanol, and was dried with nitrogen. Each of the following four steps was followed by a 3x wash in wash buffer. The reference cantilevers were incubated in 100 μg/ml cC-Ab in PBS and the active cantilevers were incubated in 100 μg/ml hC-Ab for 4 hours. Finally,

Page 6 of 26

the array was blocked with 7% ethanolamine in PBS for 1 hour and further blocked with 100 μg/ml hPEG-thiol in ethanol for 30 minutes. Low NaCl concentration PBS was used for the EDC/Sulfo-NHS experiment. The silicon array was cleaned, silanized and evaporated with gold as in the Prolinker B linking procedure. It

ip t

was then placed in 10 mM cysteamine in water overnight and washed 2x in water. A solution of 50 μg/ml cC-Ab, 55 mM Sulfo-NHS and 50 mM EDC in low concentration MES buffer was

cr

prepared and allowed to react for 15 minutes. Next, 2-mercaptoethanol was added to the solution to a final concentration of 47 mM to halt the EDC reaction, and the solution volume was doubled

us

with PB. The solution was used to functionalize the reference cantilevers for 2 hours. The array was then washed 2x in water and the process was repeated using hC-Ab to functionalize the

an

active cantilevers. Finally, the array was incubated for 15 minutes in 7% ethanolamine in PB and was washed 2x in water. A schematic representation of the functionalized cantilevers is shown

M

Figure 1.

2.5 Cantilever Measurements

d

Following the functionalization procedure, each array was placed in the Cantisens and washed 5x with PBS using 500 μl for each intake. The sample intake rate was 0.42 μl/s while the

te

intake time was 410 seconds for the glutaraldehyde and EDC/Sulfo-NHS experiments and 510

Ac ce p

seconds for the Prolinker B experiment. The sample intake was halted to avoid any deflection due to fluid flow. The flowing solution increases the rate of deflection but the increase varies from one cantilever to the next in the array. The IFN-γ concentration detected was 10 μg/ml in PBS for the glutaraldehyde and Prolinker B experiments, and 5 μg/ml in low NaCl PBS for the EDC/Sulfo-NHS experiment.

3 Theoretical Overview

3.1 ELISA Affinity Constant The affinity constants of an ELISA reflect the rate of association and disassociation of the antibody and antigen in an ELISA. The dissociation constant K d can be determined from the ELISA standard curve using linear regression and the equation:

Page 7 of 26

Y

Bmax X (1) Kd  X

where Y is the optical density, X is the antigen concentration, and Bmax is the maximum optical

ip t

density at large antigen concentration.

cr

3.2 Stress Calculation

In order to compare the cantilever measurements with those available in the literature, it

us

is useful to compute the change in surface stress. The change in deflection z of the cantilevers may be related to the change in surface stress σ through Stoney’s Equation[28]: 2

an

zE  T     (2) 31    l 

M

where E is the elastic modulus of the cantilever, υ is Poisson’s ratio, l is the length of the cantilever and T is the thickness of the cantilever.

d

3.3 Antibody-Antigen Binding Kinetics

te

The law of mass action can be used as a simple theoretical model for antibody-antigen binding[29]. The reversible reaction can be written as:

Ac ce p

F  P  C (3)

where F is the quantity of free antibody, P is the quantity of free protein and C is the quantity of antibody-protein complexes. For association constant ka and dissociation constant kd, the reversible binding reaction can be written as: dC  ka FP  k d C (4) dt

where t is time. Since the quantity of free antibody is often unknown, it is useful to replace F with A−C where A is the total quantity of antibody on the surface. The equation then becomes: dC  k a  A  C P  kd C (5) dt

Integration leads to an exponential result:

Page 8 of 26

C t  

A 1  exp  ka P  kd t  (6) kd 1 ka P

For large free antigen to antibody ratios, P effectively remains constant. Since A is also constant,

ip t

the equation can be simplified to:

C t   Ceq 1  exp  kt (7)

cr

A is the quantity of bound antigen at equilibrium, and k  k a P  k d is a time kd 1 ka P

us

where Ceq 

constant for the rate of reaction. In order to fit equation (7) to the experimental data, a linear relationship was assumed between the cantilever deflection and concentration of bound antigen

an

C(t). A similar assumption has been made in some silicon plasmon resonance measurements[30]. The new equation is:

M

Dt   Deq 1  exp  kt (8)

where D(t) is the deflection of the cantilever with time, Deq is the cantilever deflection at

te

Ac ce p

4 Results and Discussion

d

equilibrium, and k is the rate constant describing the rate of deflection.

4.1 Analysis of Linker Procedures Using ELISA The ELISA experiments were used to determine the effectiveness of the glutaraldehyde, Prolinker B and EDC/Sulfo-NHS linking procedures before they were implemented on the cantilever arrays. Effective linkers should produce a standard curve with high sensitivity, little non-specific binding, and good linearity. High sensitivity to the biomarker protein of interest, IFN-γ in this case, is critical for a good sensor, and is indicated by a large signal at all IFN-γ concentrations. This is only valid for a low rate of non-specific binding however, which is indicated by a small signal from the 0 pg/ml IFN-γ negative control chips. The experimental results are shown in Figure 2 and Table 1. The purpose of the empty well series was to serve as a positive control while the chips in 0 pg/ml IFN-γ serve as a negative control. The results show the effectiveness and reliability of the Prolinker B and glutaraldehyde linkers. The standard curves for these linkers were relatively linear, with little non-specific

Page 9 of 26

binding, and relatively large signals at all IFN-γ concentrations. The result for the EDC/SulfoNHS linking procedure was similar to that of the other linkers, with the exception of the high rate of nonspecific binding. This suggests that it is a less effective linking procedure. Kd values were determined through linear regression of equation (1) using Graphpad

ip t

Prism 5. The Kd values are all equivalent within error (Table 2). The Bmax value for Prolinker B is substantially greater than that of the other methods, indicating that it is most effective. The error

cr

in the Kd and Bmax values for the EDC/Sulfo-NHS procedure were greater than the actual values, This was caused by poorer linearity and larger variation between the two duplicates and further

us

indicates that it is less reliable than the other two linking procedures.

The detection limit of the original ELISA and the three linking procedures is shown in

an

Table 3. The detection limit was defined as the minimum average IFN-γ concentration ±2 standard deviations (SD) which does not overlap the average of the control chips without IFN-γ

4.2 Cantilever-based detection of IFN-γ

M

±2 SDs [31]. The Prolinker B procedure was more sensitive than the original ELISA.

d

The results from three representative experiments are shown in Figures 3-5. The data was recorded immediately after the IFN-γ sample intake was halted. Interestingly, the direction of

te

differential deflection of the glutaraldehyde linker is opposed to that of the other two linkers. Shu

Ac ce p

et al. suggested that the charge of the linkers may have contributed to the change in deflection direction[22]. Furthermore, the relatively long and thin glutaraldehyde linker may have allowed adjacent antibodies to simultaneously bind separate monomers of the IFN-γ homo-dimers, causing compressive stress. Additional studies are required to clarify the exact mechanism behind this effect, however.

The average difference in cantilever deflection follows an inverse exponential relationship with respect to time. Deq and k were determined by performing linear regression with Equation (8) using Graphpad Prism 5. The results are displayed in Figures 3-5 and Table 4. The k values for glutaraldhyde and Prolinker B are nearly identical while that of the EDC/Sulfo-NHS procedure is much smaller. This indicates that the rate of binding is much slower for the EDC/Shulfo-NHS procedure, making it a poorer linker. The difference in surface stress after 9 hours was 0.16±0.09, -0.11±0.04 and -0.08±0.06 N/m for glutaraldehyde, Prolinker B and EDC/Sulfo-NHS, respectively. These values are greater

Page 10 of 26

than the majority of those reported in the literature. CRP was detected at a concentration of 100 μg/ml with a surface stress of -0.089 N/m [12], human PSA was detected at a concentrations of 5 and 10 μg/ml with surface stresses of 0.036±0.003 N/m[9] and 0.02±0.01 N/m[11] respectively, and myoglobin was detected at a concentration of 50 μg/ml with a surface stress of -

ip t

0.0031±0.0006 N/m[15]. The error in [11] was read from the graph while the errors in [9] and [15] were calculated from the errors in the deflection values alone because the variation in

cr

cantilever dimensions was not given. No error values were presented in [12] aside from a small noise error of ±5 nm. The magnitude of the surface stress recorded suggests that the

us

functionalization procedures described are relatively effective.

The largest source of error in cantilever deflection was the variance in cantilever

an

deflection. Brownian noise for the cantilevers was <1 nm as calculated from [32]. The peak to peak measurement noise was <5 nm for the Prolinker B and gluteraldehyde linking procedures and <20 nm for the EDC/Sulfo-NHS linking procedure in the plateau region.

M

Good agreement between the experimental results (Figures 3-5) and equation (8) suggests a linear relationship between antibody-antigen binding and cantilever deflection/surface stress.

d

Similar deflection curves were observed in other antibody-antigen binding experiments[9, 12, 18, 24] though this pattern was particularly evident in the reports by Chen et al.[12] and Yen et

Ac ce p

te

al.[24].

5 Conclusions

IFN-γ was successfully detected using deflection cantilevers and three different linking procedures, of which the Prolinker B and gluteraldehyde linking procedures were the most reliable and effective. The greater sensitivity of the Prolinker B chips over the original ELISA procedure indicates that this linking procedure may be valuable in other immunoassays. The close correlation between antibody-antigen binding and cantilever deflection indicates that cantilever deflection and induced surface stress relate linearly to antibody-antigen binding. All three linking procedures may be beneficial for future biosensor applications as the differential surface stress indicates that they are more sensitive than those used in similar experiments. Additionally, further investigation of the demonstrated cantilever measurements may lead to the quantifiable measurement of IFN-γ and other cytokines for the diagnosis of diseases.

Page 11 of 26

Acknowledgements: The authors would like to thank the Natural Sciences and Engineering Research Council of Canada, Alberta Ingenuity and the National Research Council of Canada for financial support, and the University of Alberta Nanofab and National Institute for

ip t

Nanotechnology for technical support.

Vitae

cr

Remko van den Hurk received his B.Sc. in Engineering Physics from the University of Saskatchewan in 2007 and his M.Sc. in Electrical and Computer Engineering from the

us

University of Alberta in 2011. He is currently enrolled the Ph.D. program at the University of Alberta and his current research interests center on micro- and nano-devices for biosensors and

an

energy harvesting.

Stephane Evoy received a Ph.D. from Cornell University in 1999. He is currently an Associate Professor in the Department of Electrical and Computer Engineering at the University

M

of Alberta, as well as a Principal Investigator at the National Institute for Nanotechnology in Edmonton. Evoy's research program focuses on the development of novel micro/nanofabrication

d

technologies for the production of micro/nanoresonators for bioassays and energy harvesting applications. His current research interests also include the development of pathogen detection

te

platforms leveraging the natural advantages of bacteriophage and bacteriophage proteins. He co-

Ac ce p

edited the 2004 textbook "Introduction to Nanoscale Science and Engineering."

REFERENCES

[1] R. Craig-Schapiro, A.M. Fagan, D.M. Holtzman, Biomarkers of Alzheimer's disease, Neurobiol Dis 35 (2) (2009) 128-140. [2] D.I. Buckley, R. Fu, M. Freeman, K. Rogers, M. Helfand, C-reactive protein as a risk factor for coronary heart disease: a systematic review and meta-analyses for the U.S. Preventive Services Task Force, Ann Intern Med 151 (7) (2009) 483-495. [3] D.V. Makarov, S. Loeb, R.H. Getzenberg, A.W. Partin, Biomarkers for prostate cancer, Annu Rev Med 60 (2009) 139-151.

Page 12 of 26

[4] J.J. Graber, S. Dhib-Jalbut, Biomarkers of disease activity in multiple sclerosis, J Neurol Sci 305 (1-2) (2011) 1-10. [5] B. Jamil, F. Shahid, Z. Hasan, N. Nasir, T. Razzaki, G. Dawood, R. Hussain, Interferon gamma/IL10 ratio defines the disease severity in pulmonary and extra pulmonary tuberculosis,

ip t

Tuberculosis (Edinb) 87 (4) (2007) 279-287.

[6] H.Y. Chun, J.W. Chung, H.A. Kim, J.M. Yun, J.Y. Jeon, Y.M. Ye, S.H. Kim, H.S. Park, C.H.

cr

Suh, Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus, J Clin Immunol 27 (5) (2007) 461-466.

us

[7] P.S. Waggoner, H.G. Craighead, Micro- and nanomechanical sensors for environmental, chemical, and biological detection, Lab Chip 7 (10) (2007) 1238-1255.

an

[8] R. H. Farahi, A. Passian, L. Tetard, T. Thundat. Critical Issues in Sensor Science To Aid Food and Water Safety, ACS Nano 6 (6) (2012) 4548–4556.

[9] G. Wu, R.H. Datar, K.M. Hansen, T. Thundat, R.J. Cote, A. Majumdar, Bioassay of prostate-

M

specific antigen (PSA) using microcantilevers, Nat Biotechnol 19 (9) (2001) 856-860. [10] K.W. Wee, G.Y. Kang, J. Park, J.Y. Kang, D.S. Yoon, J.H. Park, T.S. Kim, Novel electrical

d

detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers, Biosens Bioelectron 20 (10) (2005) 1932-1938.

te

[11] M. Yue, J.C. Stachowiak, H. Lin, R. Datar, R. Cote, A. Majumdar, Label-free protein

524.

Ac ce p

recognition two-dimensional array using nanomechanical sensors, Nano Lett 8 (2) (2008) 520-

[12] C.H. Chen, R.Z. Hwang, L.S. Huang, S.M. Lin, H.C. Chen, Y.C. Yang, Y.T. Lin, S.A. Yu, Y.S. Lin, Y.H. Wang, N.K. Chou, S.S. Lu, A wireless bio-MEMS sensor for C-reactive protein detection based on nanomechanics, IEEE Trans Biomed Eng 56 (2) (2009) 462-470. [13] C. Milburn, J. Zhou, O. Bravo, C. Kumar, W.O. Soboyejo, Sensing Interactions Between Vimentin Antibodies and Antigens for Early Cancer Detection Journal of Biomedical Nanotechnology 1 (1) (2005) 9.

[14] V. Dauksaite, Lorentzen, M., Besenbacher, F., Kjems, J. , Antibody-based protein detection using piezoresistive cantilever arrays, Nanotechnology 18 (2007) 125503-125507. [15] Y. Arntz, J.D. Seelig, H.P. Lang, J. Zhang, P. Hunziker, J.P. Ramseyer, E. Meyer, M. Hegner, C. Gerber, Label-free protein assay based on a nanomechanical cantilever array, Nanotechnology 14 (2003) 86-90.

Page 13 of 26

[16] C. Grogan, R. Raiteri, G.M. O'Connor, T.J. Glynn, V. Cunningham, M. Kane, M. Charlton, D. Leech, Characterization of an antibody coated microcantilever as a potential immuno-based biosensor, Biosens Bioelectron 17 (3) (2002) 201-207. [17] R. Mukhopadhyay, V.V. Sumbayev, M. Lorentzen, J. Kjems, P.A. Andreasen, F.

ip t

Besenbacher, Cantilever sensor for nanomechanical detection of specific protein conformations, Nano Lett 5 (12) (2005) 2385-2388.

cr

[18] W. Shu, S. Laurenson, T.P. Knowles, P. Ko Ferrigno, A.A. Seshia, Highly specific labelfree protein detection from lysed cells using internally referenced microcantilever sensors,

us

Biosens Bioelectron 24 (2) (2008) 233-237.

[19] C.A. Savran, S.M. Knudsen, A.D. Ellington, S.R. Manalis, Micromechanical detection of

an

proteins using aptamer-based receptor molecules, Anal Chem 76 (11) (2004) 3194-3198. [20] Y. Lam, N.I. Abu-Lail, M.S. Alam, S. Zauscher, Using microcantilever deflection to detect HIV-1 envelope glycoprotein gp120, Nanomedicine 2 (4) (2006) 222-229.

M

[21] N. Backmann, C. Zahnd, F. Huber, A. Bietsch, A. Pluckthun, H.P. Lang, H.J. Guntherodt, M. Hegner, C. Gerber, A label-free immunosensor array using single-chain antibody fragments,

d

Proc Natl Acad Sci U S A 102 (41) (2005) 14587-14592. [22] W. Shu, E.D. Laue, A.A. Seshia, Investigation of biotin-streptavidin binding interactions

te

using microcantilever sensors, Biosens Bioelectron 22 (9-10) (2007) 2003-2009.

Ac ce p

[23] C. Montserrat, J. Tamayo, M. Nordström, A. Boisen, Low-noise polymeric nanomechanical biosensors, Appl Phys Lett 88 (11) (2006) 3. [24] Y.-K. Yen, C.-Y. Huang, C.-H. Chen, C.-M. Hung, K.-C. Wu, C.-K. Lee, J.-S. Chang, S. Lin, L.-S. Huang, A novel, electrically protein-manipulated microcantilever biosensor for enhancement of capture antibody immobilization, Sensor Actuat B-Chem 141 (2009) 498-505. [25] G. Shekhawat, S.H. Tark, V.P. Dravid, MOSFET-Embedded microcantilevers for measuring deflection in biomolecular sensors, Science 311 (5767) (2006) 1592-1595. [26] P. Dutta, J. Sanseverino, P.G. Datskos, M.J. Sepaniak, Nanostructured Cantilevers as Nanomechanical Immunosensors for Cytokine Detection, NanoBiotechnology 1 (3) (2005) 237244. [27] C.-H. Cho. Characterization of Young’s modulus of silicon versus temperature using a ‘‘beam deflection” method with a four-point bending fixture, Curr Appl Phys 9 (2009) 538-545.

Page 14 of 26

[28] G.G. Stoney, The tension of metallic films deposited by electrolysis, P R Soc London-A 32 (1909) 172–175. [29] P. Schuck, Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass

ip t

transport, Biophys J 70 (3) (1996) 1230-1249.

[30] E. Stenberg, B. Persson, H. Roos, C. Urbaniczky, Quantitative determination of surface

cr

concentration of protein with surface plasmon resonance using radiolabeled proteins, J Colloid Interf Sci 143 (2) (1991) 513-526.

us

[31] K. Saito, D. Kobayashi, M. Sasaki, H. Araake, T. Kida, A. Yagihashi, T. Yajima, H. Kameshima, N. Watanabe, Detection of human serum tumor necrosis factor-alpha in healthy

an

donors, using a highly sensitive immuno-PCR assay, Clin Chem 45 (5) (1999) 665-669. [32] H.-J. Butt, M. Jaschke, Calculation of thermal noise in atomic force microscopy,

Ac ce p

te

d

M

Nanotechnology 6 (1995) 1-7.

Page 15 of 26

Figure Captions:

Figure 1: A schematic representation of the chemical linking and blocking procedures. From left to right the results of the glutaraldehyde, Prolinker B and EDC/Sulfo-NHS linking procedures

ip t

with PEG-silane blocking are shown.

cr

Figure 2: Optical density data from the ELISA for the three linking procedures. The

us

measurements were performed in duplicate.

Figure 3: Cantilever deflection indicating the successful detection of IFN-γ using the

an

glutaraldehyde linking procedure. The diagonal, horizontal, and vertical fills represent the variance in the reference cantilever deflection, active cantilever deflection and differential

M

surface stress.

Figure 4: Cantilever deflection indicating the successful detection of IFN-γ using the Prolinker B

d

linking procedure. The diagonal, horizontal, and vertical fills represent the variance in the

te

reference cantilever deflection, active cantilever deflection and differential surface stress.

Ac ce p

Figure 5: Cantilever deflection results indicating the successful detection of IFN-γ using EDC/Sulfo-NHS linking procedure. The diagonal, horizontal, and vertical fills represent the variance in the reference cantilever deflection, active cantilever deflection and differential surface stress.

Page 16 of 26

Table Captions (Tables were attached separately for my submission) Table 1: Optical density data from the negative control chips

ip t

Table 2: Bmax and Kd values calculated from the ELISA experiments

cr

Table 3: ELISA detection limit results

Ac ce p

te

d

M

an

us

Table 4: The calculated equilibrium deflection (Deq) values and rate constants (k)

Page 17 of 26

Table 1

Optical Density 0.010, 0.011

Glutaraldehyde

0.014, 0.015

Prolinker B Empty

0.013, 0.011

Prolinker B

0.012, 0.010

EDC/Sulfo-NHS Empty

0.011, 0.012

EDC/Sulfo-NHS

0.157, 0.161

Ac ce p

te

d

M

an

us

cr

Glutaraldehyde Empty

ip t

0 pg/ml IFN-γ Control Chip

Page 18 of 26

Table 2

Bmax

Kd (×10-10)

Original ELISA

2.7±0.9

1.9±0.7

Glutaraldehyde

1.6±0.3

1.8±0.4

Prolinker B

7±1

1.4±0.2

EDC/Sulfo-NHS

2±4

2±4

Ac ce p

te

d

M

an

us

cr

ip t

Linker

Page 19 of 26

Table 3

Linker

Detection Limit (pg/ml)

Original ELISA

18±4

Glutaraldehyde

24±5

Prolinker B

8±2 50±30

Ac ce p

te

d

M

an

us

cr

ip t

EDC/Sulfo-NHS

Page 20 of 26

Table 4

Deq (nm) k (1/hours) 0.4582±

Glutaraldehyde

667.3±0.1

Prolinker B

-452.0±0.0

EDC/Sulfo-

0.0003

-406.2±0.2

0.0000 0.1766± 0.0002

0.996

1.000

0.993

Ac ce p

te

d

M

an

us

cr

NHS

0.4737±

R2

ip t

Linker

Page 21 of 26

ed

M an

us

cr

i

Figure 1

NH2

Ac

ce pt

NH2

Page 22 of 26

us 0.10

0.01

Gluteraldehyde Empty Gluteraldehyde

500

ed

50

M an

Optical Density

cr

i

Figure 2

ce pt

Optical Density

1.00

Prolinker B Empty

0.10

Prolinker B

0.01

Optical Density

Ac

50

500

0.20 EDC/Sulfo-NHS Empty EDC/Sulfo-NHS

0.02 50

500

INF-γ concentration (pg/ml)

Page 23 of 26

M an

us

cr

i

Figure 3

Deflection (nm)

0.169

0

0.000

ce pt

700

-700

-0.169

-1400

-0.337

Ac

-2100

Stress (N/m)

Reference Average Fit 0.337

ed

Active Average Difference 1400

-0.506

0

3

6

9

Time (hours)

Page 24 of 26

M an

us

cr

i

Figure 4

Deflection (nm)

ce pt

-900

-0.217

-1800

-0.433

-2700

-0.650

-3600

-0.867

Ac

-4500

Stress (N/m)

Reference Average Fit 0.000

ed

Active Average Difference 0

-1.083

0

3

6

9

Time (hours)

Page 25 of 26

M an

us

cr

i

Figure 5

Deflection (nm)

-0.072

-600

-0.144

-900

-0.217

-1200

-0.289

-1500

-0.361

ce pt

-300

Ac

-1800

Stress (N/m)

Reference Average Fit 0.000

ed

Active Average Difference 0

-0.433 0

3

6

9

Time (hours)

Page 26 of 26