Gel-Based Microchips: History and Prospects

The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.     

Microchips based on three dimensional gel cells: history and perspective

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.     

Protein adsorption modalities on polyelectrolyte multilayers

Protein adsorption on polyelectrolyte multilayers (PEMUs) was evaluated using a combination of synthetic polyelectrolytes and proteins, including serum albumin, fibrinogen, and lysozyme. Variables such as surface and protein charge, polymer hydrophobicity, and hydrophilic repulsion were introduced to probe interaction mechanisms. Quantitative analysis with reflectance Fourier transform infrared spectroscopy, optical waveguiding, and UV-vis absorption, together with qualitative information from atomic force microscopy, provided a coordinated picture for what drives protein adsorption and how the molecules are disposed on the multilayer surface. It was found that multilayers bearing a particular surface charge sorbed biomolecules if they were of opposite charge, yielding significant loadings within the bulk PEMU. Adsorption of like-charged proteins, as surface aggregates, occurred to a much lower extent, driven by nonelectrostatic forces. A diblock copolymer comprising a hydrophilic poly(ethylene oxide) block was capable of further minimizing protein adsorption as a result of hydrophilic repulsion, although none of the surfaces tested defeated protein adsorption completely. However, poly(acrylic acid) homopolymer was quite effective in this respect. A composition gradient, formed during multilayer buildup, induced a gradient in hydrophilicity through the PEMU, which is an efficient and economical method of creating a protein-resistant surface.     

Correlation between surface morphology and surface forces of protein A adsorbed on mica.

We have investigated the morphology and surface forces of protein A adsorbed on mica surface in the protein solutions of various concentrations. The force-distance curves, measured with a surface force apparatus (SFA), were interpreted in terms of two different regimens: a "large-distance" regimen in which an electrostatic double-layer force dominates, and an "adsorbed layer" regimen in which a force of steric origin dominates. To further clarify the forces of steric origin, the surface morphology of the adsorbed protein layer was investigated with an atomic force microscope (AFM) because the steric repulsive forces are strongly affected by the adsorption mode of protein A molecules on mica. At lower protein concentrations (2 ppm, 10 ppm), protein A molecules were adsorbed "side-on" parallel to the mica surfaces, forming a monolayer of approximately 2.5 nm. AFM images at higher concentrations (30 ppm, 100 ppm) showed protruding structures over the monolayer, which revealed that the adsorbed protein A molecules had one end oriented into the solution, with the remainder of each molecule adsorbed side-on to the mica surface. These extending ends of protein A overlapped each other and formed a "quasi-double layer" over the mica surface. These AFM images proved the existence of a monolayer of protein A molecules at low concentrations and a "quasi-double layer" with occasional protrusions at high concentrations, which were consistent with the adsorption mode observed in the force-distance curves.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Comparison of surface and hydrogel-based protein microchips

Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.     

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 degrees C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E(a) and the free energy of activation DeltaG(0)*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.     

Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.     

Adsorption of viral matrix protein M1 in acidic medium

Adsorption of viral matrix protein M1 on the self-assembled monolayer of carboxyhexadecanthiol molecules simulating the surface of the cell membrane was studied by surface plasmon resonance refractometry technique. It was shown that in the acidic medium (pH 4.0) the fraction of irreversibly adsorbed protein increases with time. The protein formed a monolayer on the surface in concentration range from 50 to 500 nM. It was found that the amount of the adsorbed protein increased more than 3 times in this range. An important observation is that even at the lowest concentrations of the protein its molecules totally occupied the entire surface of the substrate, and a further protein addition did not lead to its further adsorption. To explain this phenomena, it was suggested that the number of M1 bonds with the surface increases during the adsorption, which leads to spreading of the protein molecules. Apparently, this effect is caused by the intrinsic disorder of the C-domain of the protein. It is hypothesized that the disassembly of the protein-lipid envelope of the influenza virus in the acidic medium does not result from desorption of the M1, but it is caused by the weakening of protein-protein bonds.     

Immobilization of oriented protein molecules on poly(ethylene glycol)-coated Si(111)

A high-density poly(ethylene glycol) (PEG)-coated Si(111) surface is used for the immobilization of polyhistidine-tagged protein molecules. This process features a number of properties that are highly desirable for protein microarray technology: (i) minimal nonspecific protein adsorption; (ii) highly uniform surface functionality; (iii) controlled protein orientation; and (iv) highly specific immobilization reaction without the need of protein purification. The high-density PEG-coated silicon surface is obtained from the reaction of a multi-arm PEG (mPEG) molecule with a chlorine terminated Si(111) surface to give a mPEG film with thickness of 5.2 nm. Four out of the eight arms on each immobilized mPEG molecule are accessible for linking to the chelating iminodiacetic acid (IDA) groups for the binding of Cu(2+) ions. The resulting Cu(2+)-IDA-mPEG-Si(111) surface is shown to specifically bind 6x histidine-tagged protein molecules, including green fluorescent protein (GFP) and sulfotransferase (ST), but otherwise retains its inertness towards nonspecific protein adsorption. We demonstrate a particular advantage of this strategy: the possibility of protein immobilization without the need of prepurification. Surface concentrations of relevant chemical species are quantitatively characterized at each reaction step by X-ray photoelectron spectroscopy (XPS). This kind of quantitative analysis is essential in tuning surface concentration and chemical environment for optimal sensitivity in probe-target interaction.     

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 °C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E_a and the free energy of activation ΔG₀*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.     

Functional peptide microarrays for specific and sensitive antibody diagnostics

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.     

AFM and electroanalytical studies of synthetic oligonucleotide hybridization

The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.     

基于微纳技术的循环肿瘤细胞免疫检测新方法北大核心CSCD

本研究旨在探索一种高灵敏度、高特异性检测循环肿瘤细胞(circulating tumor cells, CTCs)的免疫检测新方法,以尽早地检出结直肠癌,提高该疾病的检出率。首先制备含有线性微柱结构的微芯片,通过在其表面孵育氧化石墨烯-链霉亲和素(graphite oxide-streptavidin, GO-SA)及偶联广谱一抗(antibody1, Ab₁),即上皮特异性黏附分子(epithelial cell adhesion molecule, EpCAM)单克隆抗体以捕获CTCs。运用羧基化多壁碳纳米管(carboxylated multi-walled carbon nanotubes, MWCNTs-COOH)与结直肠癌相关抗体,即特异性二抗(antibody 2, Ab₂)偶联制备抗体复合物。在捕获CTCs的微芯片上孵育该抗体复合物,构建以Ab₁-CTCs-Ab₂为主体的超级三明治结构,通过电化学工作站检测并验证其高灵敏度和高特异性。结果发现,在免疫传感器的构建中结合应用微纳技术,极大地提高了CTCs的检测灵敏度和特异性。本研究验证了该免疫传感器应用于临床血样检测的可行性,并通过该免疫传感器对结直肠癌患者外周血中CTCs进行检测和计数。结果表明,基于微纳技术的超级三明治式免疫传感器为CTCs的检测提供了新的途径,对临床工作中的疾病诊断及病情实时监控方面均具有潜在的应用价值。     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

New antibody immobilization method via functional liposome layer for specific protein assays

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.     

Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

Immunolabeling efficiency of protein A-gold complexes

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.     

Oligonucleotide-coated metallic nanoparticles as a flexible platform for molecular imaging agents

Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotide-based approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.