|
1.IntroductionDiagnosis of acute myocardial infarction (AMI) has received much attention in recent years.1 Although it is still difficult to evaluate myocardial damage in the early phase of its occurrence, an effort has been made to detect the enzymes that run early into the blood after the infarction.2 Creatine kinase and troponins have been reported to be very cardiac specific and also correlate well with the severity of the myocardial damage. However, they appear very slowly in the blood stream and need plenty of time to reach their peak.3 In spite of this, the measurement of troponins in the assessment of cardiac damage and diseases is now widespread throughout due to their longer lead times.4 For patients with severe chest pain, this troponin can provide risk stratification for the long term.5 But these tests are usually done in hospitals at a huge cost. Point-of-care testing provides a reasonable solution to the costs associated to the hospitalization of false-negative cases. A great deal of scientific research has been devoted to develop biosensors incorporated with microdevices that could provide instant results. Biosensors that have mainly focused on the analysis of proteins and enzymes have attracted considerable attention.6 These microdevices have been termed as “ideal”6 structures for analyzing and sensing these enzymes due to the reduced geometry that requires minute quantity of analytes. Microfabricated cantilevers have been proposed as preferable structures for biosensing applications.7 By applying mass on the cantilever, a change in its resonant frequency and surface stress can be monitored easily,8, 9 since the mass of troponin added in the present study is in mg per ml compared to its quantity of ng per ml in blood. Previous work10 has been done on microarrays of cantilevers to detect multiple unlabelled biomolecules at minute concentrations. The deflection of this mass-loaded cantilever beam can be evaluated with many optical detection techniques. Biomolecular interaction between target and probe molecules produces a sufficiently large force, which produces a surface stress, to bend the cantilever beam and produce motion that was detected optically.11 DNA hybridization and receptor ligand binding produces a surface stress on the cantilever that causes it bend.12 The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Any measurable disturbances in the normal behavior of a microcantilever beam are of interest in the detection of enzymes-antienzymes reaction patterns. Thus, deflection, natural frequency, or vibration modes of the cantilever beam are determined by the Rayleigh-Ritz method. In the present work, an effort has been made to analyze the behavior of PVDF-coated cantilever beams under the action of the enzymes optically. Similarly atomic force microscope (AFM) beams made of silicon were also monitored under the action of a different set of enzymes. Identification of these enzymes was also complimented by fluorescence spectroscopy (FS), and Fourier transform infrared spectroscopy (FTIR). The surface stress that is generated on the surface of the cantilevers is evaluated analytically. Scanning electron microscopy (SEM) of the enzymes on the surface was also taken. 2.Experimental Methods2.1.Optical Beam Deflection Method Using PVDF Cantilever BeamsThe experiment has been carried out in the laboratory using an optical setup. The experimental setup is presented in Fig. 1 . The setup includes a low-power laser source of and maximum wavelength of to yield a beam size of around 100 to , a spot-on complementary metal-oxide semiconductor (CMOS) camera with 3.5 million pixels, and sensitive wavelength ranging from 350 to . The position accuracy is less than with a submicron resolution of less than . It can accommodate a laser beam diameter from to . The overall detector size is a active area. Polymer-coated aluminum beams of were used such that larger amounts of reactants were deposited. The reactants were deposited using an electronic pipette that can take a minimum volume of to a maximum volume of . The error percentage was less than . 2.2.Identification of Enzymes Using Fourier Transform Infrared SpectrometryA variety of analytical tools are available to study protein conformation. Of these methods, Fourier transform infrared spectrometry (FTIR) has proven to be quite versatile.13 The versatility of FTIR is based on the long wavelengths of radiation, which minimize scattering problems. Spectral differences for proteins like troponin C (TnC) and horse radish peroxide (HRP) observed on glass samples. -square glass samples of thickness were used to detect the spectrum. Two glass samples were used, one placed on top of the other, and a gold coating was deposited on the corners between them to provide an air gap of to allow the proteins to bind with each other. 2.3.Fluorescence Spectral AnalysisFluorescence spectroscopy detected TnC-bee venom melittin (ME) binding at concentrations ranging from 2, 20, and at 1:1 complex and various other ratios. It not only determines the characteristics of single monoclonal antibodies in a short time, but also determines the characteristics of multiclonal antibodies.13 This finding is very useful for selecting monoclonal antibodies with the best application potential. Fluorescence spectroscopy observes a very large range of radiation, which minimizes scattering problems from the irradiation source. We were able to view the spectrum ranging from 200 to and found the best spectral region to be for TnC-ME binding. 3.Cantilever Model3.1.Rayleigh-Ritz ApproachThe mode shapes of the system are a complete set of orthogonal functions that are characteristics of the system. It is possible to express the deflection shape of the system in a generalized Fourier series involving mode shapes. The Rayleigh-Ritz method provides an approximate solution of the eigenvalue problem for a cantilever beam whose deflection function is of the linear form given by where is the constant coefficients or the deflection constants, and is the shape functions that are generated by the Gram-Schmidt process14 and satisfy the natural boundary conditions of the beam. The expression for Rayleigh’s quotient is the ratio of the maximum potential energy to the maximum kinetic energy of the system. It is given bywhere the kinetic energy is expressed as and is the eigenvalue. The total energy of the cantilever beam includes the potential energy, the energy due to the springs (rotational and translational), and the kinetic energies.15 Hence the maximum potential energy of the system becomes . Here, is the strain energy of the cantilever and is given by is the maximum potential energy due to the springs and is given by is the maximum kinetic energy of the beam and is given bywhere , and are the material density, cross sectional area, and the length of the beam, respectively, and is the natural frequency in radiance per second. Substituting the deflection function of Eq. 1 into the kinetic and potential energies in Eqs. 3, 4, 5, the Rayleigh quotient in Eq. 2 is given in terms of the coefficients , which minimizes into the eigenvalue equation that is given in the formwhere The natural frequency of the cantilever beam then relates to the expression given aswhere the are the eigenvalues, is the Young’s modulus of elasticity, and is the moment of inertia of the beam.3.2.Application of LoadThis droplet can be assumed to be a concentrated mass applied at the free end of the cantilever beam for simplicity in modeling, as shown in Fig. 2 . The kinetic energy of the system becomes Equation 9 can be written as follows:Equation 10 gives an expression for the evaluation of concentrated mass at the free end of the cantilever beam. Due to the addition of this extra mass, a variation in the first- and second-mode shapes can be seen in Figs. 3 and 4 .The modal analysis approach reduces the analysis time and effort, and provides results that are good engineering approximations to the actual results. 3.3.Evaluation of Surface StressWhen a cantilever beam is deformed in pure bending, we expect a uniaxial stress that is applied at three different points of the beam: at ; the bottom surface of the beam, which is compressive; at ; the middle section of the beam: and , the top surface of the beam, which is tensile. All the other stress components are identically zero.16 Shanley17 demonstrated a dependency relating surface stress with the cantilever tip deflection along the direction based on a few assumptions. The variation of is given by where is the deflection of the cantilever beam at a distance is the Young’s modulus of elasticity, is the length of the beam, and is the thickness of the beam.The tensile stress on the top surface of the cantilever beam in Fig. 5 reaches to for a deflection of . The stress variation decreases as the deflection decreases. But in the case of the bottom surface of the beam, the surface stress goes to about (see Fig. 6 ). The increase in this large value of stress is unclear at present. Experimental measurements of these structures need to be in accordance with the analytical and numerical modeling for characterizing the local stresses developed. 4.Experimental Results and Discussion4.1.Measurement of Deformation Using Biochemical ReactionEarlier experiments were carried out using optical detection techniques on AFM cantilevers on a different set of enzymes.18 The present work strongly supports the previous results with more confidence. The TnC and ME proteins were mixed in 1:1 proportions at different concentrations of 20, 25, 30, 35, and and applied over the PVDF cantilever beams. During the reaction between the enzyme and the antienzyme, the chains of enzyme molecules are opening and stretching, such that it makes the cantilever beam deflect.18 This deflection is recorded using the PSD. Once the buffer solution evaporates, the remaining solid phase on the beam arranges it self in a texture-like configuration, such that it will create a permanent stress and a strain in the cantilever. To support these assumption, SEMs of TnC-ME are shown in Figs. 7 and 8 . The binding of TnC-ME is shown in the SEM picture after complete evaporation of the buffered solution. Bee venom melittin (26-residue membrane obtained from the venom of honey bees) binds to skeletal muscle troponin C (molecular weight of 18,000 Daltons and is long) with a -dependent reaction. ME is bound to troponin C irrespective of the presence or absence of in 50-mM (potassium chloride) and 50-mM at a pH of 7.5. Measurement of cantilever deflection was performed under the circumstances for various concentrations of TnC-ME. The pattern of measurement regardless of the concentration is always almost the same and is illustrated in Figs. 9 and 10 . The displacement in micrometers at the PSD scanner in the vertical axis is given with respect to time in seconds in the horizontal axis. The figure indicates that the loading with the organic fluid solution of the beam will create a deflection of about , which is translated to the PSD in a negative (downward) displacement of the laser spot by . Further reactions will stretch the beam more due to reorganizing of the enzyme molecules to the surface of the cantilever. The reactions count for five to six more micrometers of deflection at the cantilever, which occur in about one minute. The time duration is not quite relevant, since the volumes used in the experiments were very large. Also, the beam starts moving up by with respect to the lowest previous occupied position. This motion is slow at a rate of about per second. This curving up is associated with the adhesion of the enzyme molecules on the surface of the cantilevers, a phenomenon that produces a tension in the bottom surface of the beam. This pattern of TnC-ME was compared with that of TnC-water of different concentrations. Significant differences were recorded in the pattern of displacement and amplitude. The patterns are presented in Figs. 11 and 12 , respectively. Also, troponin C binds on the surface of the cantilever, but the superficial tension produced by the troponin and water combination produces a higher deflection when compared to the troponin and melittin combination. This is relevant from the signature of reaction in Figs. 11 and 12, respectively. From the experimental setup, it was noticed that there was a time-dependent drift present in the deflection signal, and the drift in the measured signal depended almost linearly on time.19 As the concentration (mass) increases, the peak deflection also increases linearly, as shown in Fig. 13 . The higher the concentration is, the higher the deflection. Meanwhile, at higher concentrations, the duration of the reaction is high. Also, a comparison was made between the deflection of the cantilever and the time taken. The time corresponds to the time when of TnC and of ME was deposited on the cantilever surface at room temperature. The PVDF cantilever exhibited a bending of at time . As seen in Fig. 14 , the deflection rate becomes constant at when both TnC-ME evaporate completely from the surface of the cantilever. The data presented in this figure demonstrate both the high sensitivity and large dynamic range of PVDF cantilevers to TnC-ME reaction. 4.2.Spectral AnalysisIdentification of the enzymatic reactions is acquired using FTIR and fluorescence spectroscopy. Both methods share the same development level. They are not available in the microsystem scale, but eventually they could all be integrated in a chip or in hybrid architecture.20 Figures 15 and 16 indicate the comparison between ME, TnC, and TnC-ME complex due to the interaction. Due to the presence of excess , TnC undergoes a configurational change from a dumbbell structure to a globular structure21 when ME binds to TnC, and hence there is a change in fluorescence intensity, as in Fig. 16 near . In the presence of excess TnC, melittin develops a significant change in the relative absorption unit (RAU), which is seen as a positive modification of the spectrum in the tryptophan (19th amino acid residue of ME) region of around 2350 per cms, as shown in Fig. 15. Also, the change in the spectrum could be due to the removal of quenching factors, such as quenching of small molecules and amino acids. 4.3.Dynamic Characteristics for Different EnzymesThe dynamic characteristics of TnC-ME were compared to that of horse raddish persoxide (HRP) and hydrogen peroxide using the FTIR. In the presence of excess TnC, ME develops a strong positive difference spectrum in the tryptophan (Trp) region.21 It remains uncertain whether the effect results from direct involvement of Trp 19 in the region of contact, or whether it arises indirectly from the altered conformation of ME. In the case of the dynamic characteristics of these two proteins, ME binding to TnC strengthens the binding of to the troponin of the complex. Thus, the TnC-ME complex is not rigidly maintained but undergoes a transition on the binding to the low-affinity binding sites. Similarly for the case of , the RAU increases by a good 10% every , but remains steady after a period of time, showing the stability of these two sets of proteins (see Figs. 17 and 18 ). 5.ConclusionIn the present study, an effort is made to study the antigen-antibody interaction between TnC-ME and compare the dynamic characteristics with that of . Tests are performed using the optical lever method, fluorescence spectroscopy, and FTIR to understand the variation between the proteins. The dynamic behavior of cantilever beams is analyzed under the enzymatic reaction of TnC-ME. The beams are modeled accordingly to evaluate the surface stress occurring on the upper and lower surfaces of the beam. The modal analysis is done on the cantilever beam using the Rayleigh-Ritz method to find out the system’s natural frequencies and mode shapes when the system is vibrating in one of the natural frequencies. Different optical methods used in this work give a good interpretation and understanding of the enzymatic interaction. The results summarized provide strong evidence that there is an interaction between the proteins, and this information can be used in the development of biosensors that could detect AMI. AcknowledgmentThe help of Julie Bovin and Neema Chirwa from the biochemistry department at Concordia is acknowledged. This project is supported by the National Science and Engineering Research Council of Canada (NSERC). ReferencesX. Liu and
J. Wei,
“Determination of affinities and antigen epitopes of bovine cardiac troponin I (cTnl) with monoclonal antibodies by surface plasmon resonance biosensor,”
Anal. Biochem., 314 301
–309
(2003). 0003-2697 Google Scholar
K. Suzuki and
Y. Sawa,
“Early detection of cardiac damage with heart fatty acid-binding protein after cardiac operations,”
Ann. Thorac. Surg., 65 54
–58
(1997). 0003-4975 Google Scholar
V. P. Guiteras,
L. C. Pelletier,
M. G. Hernandez,
J. M. Jais,
B. R. Caitman,
G. Dupres, and
B. CG. Solymass,
“Diagnostic criteria and prognosis of preoperative myocardial infarction following coronary bypass,”
J. Thorac. Cardiovasc. Surg., 86 878
–886
(1983). 0022-5223 Google Scholar
J. R. Tate,
D. Heathcote,
J. Rayfield, and
P. E. Hickman,
“The lack of standardization of cardiac troponin I assay systems,”
Clin. Chim. Acta, 284 141
–149
(1999). 0009-8981 Google Scholar
C. W. Hamm,
J. Ravkilde,
W. Gerhardt,
P. Jorgensen,
E. Pehlim,
L. Ljungdahl,
B. Goldman, and
H. A. Katus,
“The prognostic value of serum troponin I in unstable angina,”
N. Engl. J. Med., 327 146
–150
(1992). 0028-4793 Google Scholar
P. V. A. Pamidi and
J. Wang,
“Electroanalysis and measurement of hydrazine compounds at glassy carbon electrodes coated with electropolymerized 3, -dihydroxybenaldehyde films,”
Electroanalysis, 8 244
–247
(1996). 1040-0397 Google Scholar
M. D. Antonik
N. P. D'Costa, and
J. H. Hoh,
“A biosensor on micromechanical interrogation of living cells,”
IEEE Eng. Med. Biol. Mag., 16 66
–72
(1997). https://doi.org/10.1109/51.582178 0739-5175 Google Scholar
R. Berger,
E. Delmarche,
H. P. Lang,
C. Gerber,
J. K. Gimzewki,
E. Meyer, and
H. J. Guntherodt,
“Surface stress in the self assembly of alkanethiols on gold,”
Science, 276 2021
–2023
(1997). https://doi.org/10.1126/science.276.5321.2021 0036-8075 Google Scholar
R. Raiteri,
H. J. Butt, and
M. Grattarola,
“A sensitive method to measure changes in the surface stress of solids,”
J. Colloid Interface Sci., 180 251
–260
(1996). https://doi.org/10.1006/jcis.1996.0297 0021-9797 Google Scholar
R. McKendry,
J. Zhang,
Y. Arntz, and
C. Gerber,
“Multiple label free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array,”
Proc. Natl. Acad. Sci. U.S.A., 99 9783
–9788
(2002). https://doi.org/10.1073/pnas.152330199 0027-8424 Google Scholar
W. Guanghua,
R. H. Datar,
K. M. Hansen,
T. Thundat,
R. J. Cote, and
A. Majumdar,
“Bioassay of prostate-specific antigen (PSA) using micro-cantilever,”
Nat. Biotechnol., 19 856
–860
(2001). https://doi.org/10.1038/nbt0901-856 1087-0156 Google Scholar
J. Fritz,
M. K. Baller,
H. P. Lang,
H. Rothuizen,
P. Vettiger,
E. Meyer,
H. Guntherodt,
C. Gerber, and
J. K. Gimzewski,
“Translating biomolecular recognition into nanomechanics,”
Science, 288 316
–318
(2000). https://doi.org/10.1126/science.288.5464.316 0036-8075 Google Scholar
P. I. Harris,
“Fourier transform infrared studies of peptides: Potentials and pitfalls,”
ACS Symp. Ser., 750 54
–95
(2000). 0097-6156 Google Scholar
R. B. Bhat,
“Natural frequencies of rectangular plates using characterization orthogonal polynomials in Rayleigh-Ritz method,”
J. Sound Vib., 102 493
–497
(1985). 0022-460X Google Scholar
G. Rinaldi,
M. Packirisamy, and
I. G. Stiharu,
“Experimental investigation on the dynamics of MEMS structures,”
Proc. SPIE, 5455 66
–73
(2004). 0277-786X Google Scholar
V. T. Srikar,
K.. S. Anna,
M. U. Selim,
B. B. Goldberg, and
S. M. Spearing,
“Micro-Raman measurement of bending stresses in micromachined silicon flexures,”
J. Microelectromech. Syst., 12 779
–787
(2003). https://doi.org/10.1109/JMEMS.2003.820280 1057-7157 Google Scholar
F. R. Shanley,
(1957) Google Scholar
J. Amritsar,
I. Stiharu, and
M. Packirisamy,
“Micro electro mechanical systems (MEMS) for enzymatic detection,”
Proc. SPIE, 5455 101
–109
(2004). 0277-786X Google Scholar
P. G. Datskos and
I. Sauers,
“Detection of 2-mercaptoethanol using gold-coated micromachined cantilevers,”
Sens. Actuators B, B61 75
–82
(1999). https://doi.org/10.1016/S0925-4005(99)00251-8 0925-4005 Google Scholar
J. Amritsar,
I. Stiharu, and
M. Packirisamy,
“MOEMS for bio-enzymatic detection,”
Can. Inst. Photon. Innov. (CIPI), 3
(1), 25
–27
(2005) Google Scholar
J. Amritsar,
I. Stiharu, and
M. Packirisamy,
“Enzymatic detection of troponin C and melittin bee,”
Proc. SPIE, 5705 40
–51
(2005). 0277-786X Google Scholar
|