Solvent-programmed microchip open-channel electrochromatography

Open-channel electrochromatography in combination with solvent programming is demonstrated using a microchip device. Channel walls were coated with octadecylsilanes at ambient temperatures, yielding stationary phases for chromatographic separations of neutral dyes. The electroosmotic flow after coating was sufficient to ensure transport of all species and on-chip mixing of isocratic and gradient elution conditions with acetonitrile-buffer mixtures. Chips having different channel depths between 10.2 and 2.9 μm were evaluated for performance, and van Deemter plots were established. Channel depths of about 5 μm were found to be a good compromise between efficiency and ease of operation. Isocratic and gradient elution conditions were easily established and manipulated by computer-controlled application of voltages to the terminals of the microchip. Linear gradients with different slopes, start times, duration times, and start percentages of organic modifier proved to be powerful tools to tune selectivity and analysis time for the separation of a test mixture. Even very steep gradients still produced excellent efficiencies. Together with fast reconditioning times, complete runs could be finished in under 60 s.     

Chip-based bioassay using bacterial sensor strains immobilized in three-dimensional microfluidic network

A whole-cell bioassay has been performed using Escherichia coli sensor strains immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the silicon chip, and thus, a three-dimensional microfluidic network is constructed in the assembly. Bioluminescent sensor strains mixed with agarose are injected into the channels on one of the two PDMS layers and are immobilized in the microwells by gelation. Induction of the firefly luciferase gene expression in the sensor strains can be easily carried out by filling the channels on the other layer with sample solutions containing mutagen. Bioluminescence emissions from each well are detected after injection of luciferin/ATP mixtures into the channels. In this assay format using two multichannel layers and one microwell array chip, the interactions between various types of samples and strains can be monitored at each well on one assembly in a combinatorial fashion. Using several genotypes of the sensor strains or concentrations of mitomycin C in this format, the dependence of bioluminescence on these factors was obtained simultaneously in the single screening procedure. The present method could be a promising on-chip format for high-throughput whole-cell bioassays.     

Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device

We have developed a microfluidic device for on-chip monitoring of cellular reactions. The device consists of two primary analytical functions: control of cell transport and immobilization, and dilution of an analyte solution to generate a concentration gradient. In this device, a dam structure in parallel to the fluid flow was constructed for docking and alignment of biological cells, which allows the fragile cells to move in the microfluidic channels and to be immobilized with controllable numbers in desired locations. The cells docked on the parallel dam structure are exposed to minimal stress caused by fluidic pressure. Additionally, a network of microfluidic channels was designed to generate a concentration gradient by controlled fluid distribution and diffusive mixing. An analyte solution could be diluted to different gradients as a function of distance along the dam. We used the ATP-dependent calcium uptake reaction of HL-60 cells as a model for on-chip measurement of the threshold ATP concentration that induces significant intracellular calcium signal. The results have demonstrated the feasibility of using the microchip for real-time monitoring of cellular processes upon treatment of a concentration gradient of a test solution. The integration of cell manipulation and solution manipulation on a microchip allows the measurement of concentration-dependent biological responses within a confined microscale feature.     

Optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis

We investigated the optimal surface chemistry of peptide immobilization for on-chip phosphorylation analysis. In our previous study, we used a heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) to immobilize cysteine-terminated peptides on an amine-modified gold surface. The study revealed that the phosphorylation efficiency and rate were low (only 20% at 2 h) comparing with the reaction in solution. In this study, to improve the phosphorylation efficiency, the kinase substrates were immobilized via poly(ethylene glycol) (PEG), a flexible, hydrophilic polymer. An improvement in cSrc phosphorylation was achieved (60% at 1 h) from using a PEG-inserted peptide and SSMCC. However, no phosphorylation could be detected when the peptide was immobilized with a PEG-containing cross-linker. Fluorescence-labeled peptide studies revealed that the use of longer cross-linkers resulted in lower immobilization density. We considered that the flexible PEG linker was preferable to secure high phosphorylation efficiency for the immobilized peptide, probably due to the improvement of cSrc accessibility and peptide mobility, but the immobilization protocol is critical for keeping high density of the peptide immobilization. In addition, such an accelerating effect of PEG linker against on-chip phosphorylation of an immobilized peptide may depend on kinase structures or the position of the active center, because no improvement of on-chip peptide phosphorylation was observed in protein kinase A. However, PEG linker also did not suppress the phosphorylation in protein kinase A. Thus, we concluded that SSMCC and PEGylated peptide will be a good combination for the surface chemistry of on-chip phosphorylation in peptide array.     

A general method for patterning gradients of biomolecules on surfaces using microfluidic networks

This report outlines a general method for the fabrication of immobilized gradients of biomolecules on surfaces. This method utilizes a microfluidic network that generates a gradient of avidin in solution and immobilizes this protein on the surface of glass or poly(dimethylsiloxane) by physical adsorption. The immobilized gradient of avidin is then translated into gradients of biotinylated ligands (e.g., small molecules, oligomers of DNA, polysaccharides) using the specific interaction between biotin and avidin. This method can also generate immobilized gradients of certain proteins and artificial polymers by a direct transfer of gradients from solution onto the surface. The major advantage of this method is that almost any type of molecule can, in principle, be immobilized in a well-defined surface gradient of arbitrary shape with dimensions of a few micrometers to a few centimeters. It is possible to tailor the precise shapes of gradients on surfaces from gradients in solution, either kinetically or competitively. Kinetic methods rely on controlling the time that the surface is exposed to the gradient in solution: when a single protein adsorbs from solution, the amount that adsorbs depends both on its concentration in solution and on the time allowed for adsorption. Competitive methods rely on exposure of the surface to a complementary gradient of two proteins in solution (In these experiments, the sum of the concentrations of the proteins in solution is independent of positions although the concentration of each, individually, depends on the position. In this procedure, the relative amount of each protein, at saturation on the surface, depends only on its concentration.).     

Microscale isoelectric fractionation using photopolymerized membranes

In this work, we introduce microscale isoelectric fractionation (μIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. μIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.     

Gradient chromatofocusing. versatile pH gradient separation of proteins in ion-exchange HPLC: characterization studies

A new chromatofocusing technique called gradient chromatofocusing is characterized. Gradient chromatofocusing generates linear pH gradients on anion-exchange columns with inexpensive low molecular mass buffer components via HPLC gradient mixing. Gradient chromatofocusing results are compared with that of conventional chromatofocusing in the chromatography of several proteins on a Mono P column, including beta-lactoglobulin A and B, ovalbumin, BSA, and conalbumin. Gradient chromatofocusing shows superior performance, with resolution increases greater than 3-fold being realized for the entire protein mixture and up to 25-fold for a particular protein pair. This performance superiority arises from inherent advantages in the gradient chromatofocusing technique in optimizing conditions pertinent to separation, including buffer concentration and pH gradient slope. These resolution gains arise from both increases in separation factor and decreases in peak width achieved with the pH gradient chromatofocusing technique through the manipulation of buffer concentration and the pH gradient profile. Gradient chromatofocusing is also compared with conventional NaCl gradient ion-exchange chromatography using the same Mono P column, demonstrating 3-fold resolution gains, resulting from a 3-fold decrease in peak width. The present work demonstrates the significantly improved performance that gradient chromatofocusing has in protein separations compared to other ion-exchange chromatographic techniques. Mechanisms for the various effects are discussed.     

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.     

Size exclusion chromatography of microliter volumes for on-line use in low-pressure microfluidic systems

We have developed a new cartridge format for on-line size exclusion processing in low-pressure, portable microfluidic devices. The described system allows size exclusion chromatography of microliter volumes (termed muL-SEC) to be performed with ready integration in complex protocols for continuous-flow sample processing and analysis. The refillable cartridge format was employed for the preparation of Bacillus subtilus spores lysed in the presence of a strong reducing agent. While the reducing agent is known to interfere with subsequent fluorescent labeling of the solubilized proteins, the described continuous-flow size exclusion processing allowed complete isolation of interferences from a 10-muL sample in 70 s. Following efficient labeling, the protein sample was injected and separated on-chip using gel electrophoresis. To increase the resolution, speed, and sample capacity of buffer exchange under low-pressure operation (40 psi), parameters such as the size exclusion resin, load volumes, flow rates, buffer composition, and cartridge geometry were optimized and are presented here. The muL-SEC analysis is compatible with automated sample preparation for microfluidic systems and has resulted in significantly increased analysis speed and throughput over benchtop methods. The presented technique has the potential to improve capabilities such as buffer exchange, size fractionation, and high-abundance protein removal-steps that are frequently required prior to on-chip, point-of-care, and mass spectrometric analyses.     

pH dependent uptake of loperamide across the gastrointestinal tract: an in vitro study

Loperamide is a peripherally acting antidiarrheal opioid with some affinity for P-glycoprotein (P-gp). One of the main reasons for its lack of central nervous system (CNS) activity is a combination first-pass metabolism and P-gp-mediated efflux preventing brain penetration. It was assumed that P-gp would also have a similar effect at the intestinal tract, limiting loperamide systemic absorption. However, previous in vitro studies had not determined loperamide flux using pH gradients present in the intestinal tract. Hence, our aim was to determine the influence of pH gradient conditions on the gastrointestinal uptake of loperamide, including any changes to its P-gp-mediated efflux. METHODS: Cellular uptake and transcellular transport were determined after exposure to various concentrations of loperamide (2-50 microM) with and without the presence of active efflux protein inhibitors. Loperamide was detected at 214 nm using high-performance liquid chromatography (HPLC) protocols. RESULTS: Bidirectional transport studies of 10 microM loperamide with a pH 6.0/7.4 apical (Ap)-to-basolateral (Bas) gradient showed efflux to be 17-fold higher than influx (10 ng/cm2/min Bas-->Ap compared to 0.6 for Ap-->Bas). This differential was much greater than when examined at pH 7.4/7.4 (only two-fold higher). The potent P-gp inhibitor, PSC-833, had only a moderate effect at blocking loperamide efflux under pH gradient conditions, yet could equilibrate bidirectional transport at pH 7.4. This suggested the presence of significant P-gp independent mechanisms, preventing loperamide access to the basolateral chamber. Amiloride and 5-(N-ethyl-N-isopropyl) amiloride had some effect on reducing efflux, hence the Na(+)--H(+) antiporter may have some involvement. Accumulation of loperamide into Caco-2 cells reduced almost 70% at pH 6.0 compared to pH 7.4, yet P-gp was always able to approximately double the equilibrium concentration in the cells within a defined pH study. This showed that P-gp was not affected by pH conditions. CONCLUSIONS: P-gp-mediated efflux of loperamide is supplemented under pH gradient conditions. Hence, drugs used to decrease acid secretion in the stomach could result in higher plasma loperamide levels based on our in vitro system reflecting the in vivo environment. The addition of a P-gp inhibitor could potentially further increase the gastrointestinal absorption of loperamide.     

Method for microfluidic whole-chip temperature measurement using thin-film poly(dimethylsiloxane)/rhodamine B

A novel method is presented for on-chip temperature measurements using a poly(dimethylsiloxane) (PDMS) thin film dissolved with Rhodamine B dye. This thin film is sandwiched between two glass substrates (one of which is 150 microm thick) and bonded to a microchannel molded in a PDMS substrate. Whole-chip (liquid and substrate) temperature measurements can be obtained via fluorescent intensity visualization. For verification purposes, the thin film was tested with a tapered microchannel subjected to Joule heating, with resulting axial temperature gradients comparing well with numerical simulations. Errors induced by the definite film thickness are discussed and accounted for during experimental and analytical analysis. Alternative validation using the traditional in-channel Rhodamine B injection method was also attempted. The thin film has several advantages over traditional methods. First, false intensity readings due to adsorption and absorption of Rhodamine B into PDMS channels are eliminated. Second, whole-chip temperature measurements are possible. Third, separation of working liquid from Rhodamine B dye prevents possible electrophoresis effects.     

Rapid and reversible generation of a microscale pH gradient using surface electric fields

We report a method for the rapid and reversible generation of microscale pH gradients using a spatially varied electric field. A linear gradient in electrochemical potential is produced on an electrode surface consisting of a platinum catalyst layer on indium-tin oxide-coated glass by the application of two different potential values at spatially distinct surface locations. The resulting potential gradient drives the oxidation and reduction of water at different rates along the surface, as dictated by the local applied potential. A nonuniform distribution of pH in the neighboring solution results due to the variation in surface reaction rates. The extent and magnitude of the pH gradient can be controlled by the appropriate selection of applied potential values. In addition, the gradient can be rapidly turned on or off and reversibly switched between various profiles. The size of the pH gradient can be readily modified by changing the dimensions of the electrode and contact pads to allow integration into chip-scale devices. Characteristics of the pH gradient are described, including experimental and theoretical evidence of significant improvement in time response over competing methods for the generation of microscale pH gradients.     

DNA separation methodology based on charge neutralization in a polycationic gel matrix

A novel method for separation of DNA fragments is here reported, based on migrating the polyanionic DNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated under two working conditions: either against a gradient of positive charges, to allow the various DNA fragments to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization, or in a plateau gel (i.e., in a gel containing a constant level of positive charges from anode to cathode). In this last case, separation is still obtained due to differential charge modulation of the various DNA fragments. In the 100-1000-bp length, it is shown that separation can be obtained even for fragments differing in length by <0.5%, as shown in the splitting of a 656- and 659-bp doublet, that could not be resolved by conventional polyacrylamide gels. In the 10-100-bp range, it is shown that the present method can resolve single nucleotide polymorphisms, i.e. fragments of identical number of nucleotides but differing by one base substitution. In this last case, separations are obtained only in gradient gels containing a much steeper gradient of charges (0-20 mM Immobiline pK 10.3 and pK 12, as opposed to gradients of only 2-4 mM positive charges for larger size fragments). This novel methodology represents a marked improvement over existing techniques and appears to hold promises for applications in diverse fields, such as molecular biology, forensic medicine, and genetic screening.     

Isoelectric focusing in a microfluidically defined electrophoresis channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.     

Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: horseradish peroxidase immobilized on magnetic beads

Horseradish peroxidase was immobilized on the magnetic poly(glycidylmethacrylate-co-methylmethacrylate) (poly(GMA-MMA)), via covalent bonding and used for the treatment of phenolic wastewater in continuous systems. For this purposes, horseradish peroxidase (HRP) was covalently immobilized onto magnetic poly(GMA-MMA) beds using glutaraldehyde (GA) as a coupling agent. The maximum HRP immobilization capacity of the magnetic poly(GMA-MMA)-GA beads was 3.35 mg g(-1). The immobilized HRP retained 79% of the activity of the free HRP used for immobilization. The immobilized HRP was used for the removal of phenol and p-chlorophenol via polymerization of dissolved phenols in the presence of hydrogen peroxide (H(2)O(2)). The effect of pH and temperature on the phenol oxidation rate was investigated. The results were compared with the free HRP, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP. The optimum pH value for free and immobilized HRP was observed at pH 7.0. The optimum temperature for phenols oxidation with immobilized HRP was between 25 and 35 degrees C and the immobilized HRP has more resistance to temperature inactivation than that of the free form. Finally, the immobilized HRP was operated in a magnetically stabilized fluidized bed reactor, and phenols were successfully removed in the enzyme reactor.     

General microarray technique for immobilization and screening of natural glycans

We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.     

Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays

In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.     

Equilibrium gradient methods with nonlinear field intensity gradient: a theoretical approach

Equilibrium gradient methods belong to a family of separation techniques in which analytes are forced to unique equilibrium points by a force gradient and a counter force along the separation pathway. The basic theory for equilibrium gradient methods where the force gradient is induced by a field gradient is developed in this paper. The results indicate that peak capacity can be dynamically improved by using a nonlinear field-intensity gradient in which the first section is steep, and the following section is shallow. Using electromobility focusing (EMF) as an example, a separation model is established. EMF is an equilibrium gradient method that uses an electric field intensity gradient to induce a force gradient on charged analytes, such as proteins, and a constant hydrodynamic flow as an opposing force. Equations relating operating parameters with separation performance are given. Although simulation results show that a peak capacity of over 10,000 is theoretically possible using a single channel in a separation time just under 2 months, if 100 parallel separation units are utilized in an array format under the same operating conditions, the same peak capacity can be obtained in just over 12 h.     

Immobilization of epidermal growth factor on titanium and stainless steel surfaces via dopamine treatment

Titanium and stainless steel were modified with dopamine for the immobilization of biomolecules, epidermal growth factor (EGF). First, the treatment of metal surfaces with a dopamine solution under different pH conditions was investigated. At higher pH, the dopamine solution turned brown and formed precipitates. Treatment of the metals with dopamine at pH 8.5 also resulted in the development of brown color at the surface of the metals. The hydrophobicity of the surfaces increased after treatment with dopamine, independently of pH. X-ray photoelectron spectroscopy revealed the formation of a significant amount of an organic layer on both surfaces at pH 8.5. According to ellipsometry measurements, the organic layer formed at pH 8.5 was about 1000 times as thick as that formed at pH 4.5. The amount of amino groups in the layer formed at pH 8.5 was also higher than that observed in the layer formed at pH 4.5. EGF molecules were immobilized onto the dopamine-treated surfaces via a coupling reaction using carbodiimide. A greater amount of EGF was immobilized on surfaces treated at pH 8.5 compared with pH 4.5. Significantly higher growth of rat fibroblast cells was observed on the two EGF-immobilized surfaces compared with non-immobilized surfaces in the presence of EGF. The present study demonstrated that metals can become bioactive via the surface immobilization of a growth factor and that the effect of the immobilized growth factor on metals was greater than that of soluble growth factor.     

Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization

We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.