Identification of C10 biotinylated camptothecin (CPT-10-B) binding peptides using T7 phage display screen on a QCM device

A peptide sequence that can bind to camptothecin (CPT), a natural cytotoxic compound, was screened for using a T7 phage display system combined with a cuvette type quartz crystal microbalance (QCM) device. In this screen, after only 10 min of monitoring of the interaction between injected T7 phage pool with immobilized C10 biotinylated CPT (CPT-10-B) on a gold electrode surface, six different kinds of phage (A–F) were identified as judged by the size of PCR product on agarose gel electrophoresis. Injection of each single phage (A–E) pool individually caused a frequency decrease, suggesting interaction with the immobilized CPT-10-B. In addition, the peptide sequence displayed on phages A–C is consistent with chemical and biological studies of the interaction of CPTs with topoisomerase I (TopI), human E prostanoid receptor third cytoplasmic polypeptide, and a series of esterases. The efficacy of T7 phage display screening for small molecules on QCM devices, target discovery from primary peptide sequence, and application of this strategy to various drug-like small molecules are discussed.     

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.     

Synthesis of tethered-polymer brush by atom transfer radical polymerization from a plasma-polymerized-film-coated quartz crystal microbalance and its application for immunosensors

This study synthesizes a tethered surface-grafted poly(acrylic acid) with quartz crystal microbalance (QCM) surfaces and provides detailed analysis of their properties and application. A tethered polyelectrolyte brush of poly(acrylic acid) is generated by first covering the substrate with a plasma-polymerized allyl alcohol (pp-AA) film, changing the polymerization initiators (bromination), and then grafting through atom transfer radical polymerization (ATRP) of tert-butyl acrylate (t-BA); these initiators are immobilized on a surface and exposed to a monomer. Finally, we convert the poly(t-BA) brush into poly(acrylic acid) through hydrolysis. We use the QCM technique to measure configuration change of the tethered poly(acrylic acid) grafted chains with two different degrees of polymerization (DP=50,200) in aqueous solutions at three different pH values (4.0, 4.8, and 5.4). The tethered poly(acrylic acid) grafted QCM shows that repeatable frequency responses are induced by pH change of solution. These frequency responses of large DP for pH are 20 times larger than responses of lower DP for pH. The frequency response of antibody immobilization on tethered poly(acrylic acid) grafted QCM (DP=200) and its frequency response of immunoreaction are 10 times larger than conventional immobilization methods by cysteamine with glutalaldehyde coupling of the antibody. The tethered poly(acrylic acid) grafted QCM can increase the frequency response for pH, the immobilization amount of antibody, and immunosensor response.     

Change in rigidity in the activated form of the glucose/galactose receptor from Escherichia coli: a phenomenon that will be key to the development of biosensors

Recently a periplasmic glucose/galactose binding protein, GGRQ26C, immobilized on a gold surface has been used as an active part of a glucose biosensor based on quartz microbalance technique. However the nature of the glucose detection was not clear. Here we have found that the receptor protein film immobilized on the gold surface increases its rigidity when glucose is added, which explains the unexpected detection signal. To study the rigidity change, we developed a new fast and simple method based on using atomic force microscopy (AFM) in tapping mode. The method was verified by explicit measurements of the Young's modulus of the protein film by conventional AFM methods. Since there are a host of receptors that undergo structural change when activated by ligand, AFM can play a key role in the development and/or optimization of biosensors based on rigidity changes in biomolecules.     

Nonspecific hydrophobic interactions of a repressor protein, PhaR, with poly[(R)-3-hydroxybutyrate] film studied with a quartz crystal microbalance

The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly[(R)-3-hydroxybutyrate] (P(3HB)) were investigated quantitatively by using a quartz crystal microbalance (QCM) technique. Adsorption of PhaR onto a melt-crystallized film of P(3HB) (cr-P(3HB)) was detected as a negative frequency shift of the QCM. The time course of the frequency changes observed for PhaR adsorption was composed of a quick frequency decrease at an initial stage and a subsequent slower frequency decrease for several hours, indicating multilayered adsorption of PhaR molecules onto cr-P(3HB). The initial rapid adsorption, which corresponds to direct adsorption of PhaR molecules onto a bare surface of cr-P(3HB), was a diffusion-controlled process. Strong interactions between PhaR and cr-P(3HB) were also observed as apparently irreversible adsorption. The comparative QCM measurement of PhaR adsorption onto various types of polymers with different aliphatic chemical structures revealed that PhaR was adsorbed onto the surfaces of polymers, including cr-P(3HB), mainly by nonspecific hydrophobic interactions. These results illustrate the high affinity and low specificity for adsorption of PhaR to P(3HB).     

Piezoelectric olfactory biosensor: ligand specificity and dose-dependence of an olfactory receptor expressed in a heterologous cell system

An olfactory receptor protein of rats, I7, was expressed on the surface of human embryonic kidney (HEK)-293 cells. For targeting and detecting the protein, rho-tag import sequence was fused with the I7 protein. The olfactory receptor was expressed on the plasma membrane of HEK-293 cells, and stable cell lines regulated by an inducer were obtained. The expression on the cell surface was confirmed by immunocytochemical and Western blotting methods, and the binding of specific odorant molecules to the olfactory receptor was measured using quartz crystal microbalance (QCM). The results for QCM coated with cells containing the olfactory receptor showed that the expressed protein I7 strongly interacted with octyl aldehyde (octanal), which is an odorant specific to the I7 protein. Several other odorants were tested, and the results showed that I7 interacted differently with them. The QCM response to the serial concentrations of octyl aldehyde showed that the response is dose dependent. All these results indicate that the I7 receptor protein expressed on the surface of the heterologous cell system is sensitive to the specific odorant and can be used for the quantitative measurement of the odorant.     

Concentration dependence of IgG-protein A affinity studied by wireless-electrodeless QCM

The binding affinity between human immunoglobulin G (IgG) and protein A was studied by the homebuilt wireless-electrodeless quartz crystal microbalance (QCM). Protein A was immobilized on the electrodeless AT-cut quartz plate of 0.05 mm thick and its fundamental resonance frequency near 34 MHz was measured by a noncontacting manner using a line antenna. The vibrational analysis was performed to ensure higher sensitivity of the electrodeless QCM. A flow-cell system was fabricated to continuously measure the resonance frequency during the injection sequence of the IgG solutions with concentrations of 1-20,000 ng/mL. The exponential frequency changes were recorded to determine the affinity based on the Langmuir kinetics. The equilibrium constant K(A) significantly varied between 6 x 10(6) and 6 x 10(10) M(-1), depending on the IgG concentration, which is attributed to various formations of IgG-protein A complexes.     

Frequency dependent and surface characterization of DNA immobilization and hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued.     

Quartz crystal microbalance bioaffinity sensor for biotin based on mixed self-assembled monolayers and metastable molecular complex receptor

A quartz crystal microbalance (QCM) sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer (SAM) of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM, and enhances the amount of the immobilized molecules on the QCM, as a longer "space arm" in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017-1.67 microg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.     

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.     

Surface functionalization of polypyrrole film with glucose oxidase and viologen

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).     

Studies of the binding and signaling of surface-immobilized periplasmic glucose receptors on gold nanoparticles: a glucose biosensor application

Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.     

Acoustic wave immunosensing of a meningococcal antigen using gold nanoparticle-enhanced mass sensitivity

Bacterial meningitis is an infection of the thin membranes covering the brain and spinal cord by a number of microorganisms including Neisseria meningitidis, which can lead to permanent neurological damage in the event of late diagnosis. Given the quick onset and severity of the disease, there is a clear need for a rapid, sensitive and specific diagnostic technique. Here, we describe the development and evaluation of an acoustic wave sensor, the quartz crystal microbalance (QCM), as a rapid immunosensor employing antibodies against the cell surface outer membrane protein 85 (OMP85) of N. meningitidis as an immobilized selective layer. These antibodies were directionally orientated as receptors by thin film deposition of structured polyvinylidene fluoride and Protein A. The sensitivity of this QCM immunosensor was further increased by conjugation of the OMP85 antigen to 50 nm gold nanoparticles providing reproducible detection of the target down to 300 ng/mL. Subsequent treatment of the QCM surface with an acidic glycine solution regenerated the immunosensor allowing each crystal to be used several times.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic beta-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin-biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.     

Piezoelectric biosensor using olfactory receptor protein expressed in Escherichia coli

An olfactory receptor protein of C. elegans, ODR-10, was expressed in Escherichia coli as a fusion protein, with GST and 6x His-tag. The expression of the target protein was analyzed by SDS-PAGE and Western blot, and was confirmed to be expressed at the membrane fraction of the host E. coli. The surface of a quartz crystal microbalance (QCM) was coated with crude membrane extracts, containing the expressed receptor protein, and the interaction between the olfactory receptor and various odorant molecules examined. Compared with other odorants, diacetyl (2,3-butanedione), known as a natural ligand for the ODR-10 receptor, interacted most strongly with the expressed protein. Various concentrations of diacetyl were applied to the expressed ODR-10 receptor, and the response of the QCM showed a linear relationship to the logarithmic value of the odorant concentration. This piezoelectric biosensor system, using olfactory receptor proteins expressed in E. coli, can be used in diagnostics, toxic chemical detection and the quality control of food.     

A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Simultaneous monitoring of electroformation of phospholipid vesicles by quartz crystal microbalance and optical microscopy

The electroformation of giant vesicles from 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) and optical microscopy, simultaneously using a novel sample cell design. A gold-coated QCM crystal was used as one of the electrodes and an Indium–tin-oxide (ITO)-coated glass slide was used as the second electrode for electroformation. Increases in the frequency and decreases in the dissipation were observed immediately upon voltage application between the two electrodes, indicating the loss of lipid from the QCM surface. Concurrently, we observed vesicles on the QCM electrode surface by differential interference contrast (DIC)-optical microscopy. The lipid-coated substrates were measured with AFM at various stages in the electroformation, and a significant change in the morphology of the lipid film was observed. Ellipsometry was used to find the average thickness of lipid film. The QCM data were fitted to a viscoelastic model to determine the viscoelastic properties and time dependence of the film thickness. All methods used to determine film thickness give values in reasonable quantitative agreement. Differences between the methods are consistent with what one might expect due to what is actually measured in the individual techniques. The comparison between mass loss and observed vesicles suggest that the vesicles formed are first localized to the substrate and then slowly released into the solution. By comparing the mass lost from the lipid film, to the total surface area of lipid vesicles observed, it is apparent that only a relatively small fraction of the lipid goes into the production of unilamellar vesicles with sizes detectable with optical microscopy.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.