Particle sensing with confined optical field enhanced fluorescence emission (Cofefe).

We describe the development and performance of a new type of optical sensor suitable for registering the binding/dissociation of nanoscopic particles near a gold sensing surface. The method shares similarities with surface plasmon resonance microscopy but uses a completely different optical signature for reading out binding events. This new optical read-out mechanism, which we call confined optical field enhanced fluorescence emission (Cofefe), uses pulsed surface plasmon polariton fields at the gold/liquid interface that give rise to confined optical fields upon binding of the target particle to the gold surface. The confined near-fields are sufficient to induce two-photon absorption in the gold sensor surface near the binding site. Subsequent radiative recombination of the electron-hole pairs in the gold produces fluorescence emission, which can be captured by a camera in the far-field. Bound nanoparticles show up as bright confined spots against a dark background on the camera. We show that the Cofefe sensor is capable of detecting gold and silicon nanoparticles, as well as polymer nanospheres and sub-µm lipid droplets in a label-free manner with average illumination powers of less than 10 µW/µm2.

Measuring melittin uptake into hydrogel nanoparticles with near-infrared single nanoparticle surface plasmon resonance microscopy.

This paper describes how changes in the refractive index of single hydrogel nanoparticles (HNPs) detected with near-infrared surface plasmon resonance microscopy (SPRM) can be used to monitor the uptake of therapeutic compounds for potential drug delivery applications. As a first example, SPRM is used to measure the specific uptake of the bioactive peptide melittin into N-isopropylacrylamide (NIPAm)-based HNPs. Point diffraction patterns in sequential real-time SPRM differential reflectivity images are counted to create digital adsorption binding curves of single 220 nm HNPs from picomolar nanoparticle solutions onto hydrophobic alkanethiol-modified gold surfaces. For each digital adsorption binding curve, the average single nanoparticle SPRM reflectivity response, ⟨Δ%RNP⟩, was measured. The value of ⟨Δ%RNP⟩ increased linearly from 1.04 ± 0.04 to 2.10 ± 0.10% when the melittin concentration in the HNP solution varied from zero to 2.5 µM. No change in the average HNP size in the presence of melittin is observed with dynamic light scattering measurements, and no increase in ⟨Δ%RNP⟩ is observed in the presence of either FLAG octapeptide or bovine serum albumin. Additional bulk fluorescence measurements of melittin uptake into HNPs are used to estimate that a 1% increase in ⟨Δ%RNP⟩ observed in SPRM corresponds to the incorporation of approximately 65000 molecules into each 220 nm HNP, corresponding to roughly 4% of its volume. The lowest detected amount of melittin loading into the 220 nm HNPs was an increase in ⟨Δ%RNP⟩ of 0.15%, corresponding to the absorption of 10000 molecules.

Surface Enzyme Chemistries for Ultrasensitive Microarray Biosensing with SPR Imaging.

The sensitivity and selectivity of surface plasmon resonance imaging (SPRI) biosensing with nucleic acid microarrays can be greatly enhanced by exploiting various nucleic acid ligases, nucleases, and polymerases that manipulate the surface-bound DNA and RNA. We describe here various examples from each of these different classes of surface enzyme chemistries that have been incorporated into novel detection strategies that either drastically enhance the sensitivity of or create uniquely selective methods for the SPRI biosensing of proteins and nucleic acids. A dual-element generator-detector microarray approach that couples a bioaffinity adsorption event on one microarray element to nanoparticle-enhanced SPRI measurements of nucleic acid hybridization adsorption on a different microarray element is used to quantitatively detect DNA, RNA, and proteins at femtomolar concentrations. Additionally, this dual-element format can be combined with the transcription and translation of RNA from surface-bound double-stranded DNA (dsDNA) templates for the on-chip multiplexed biosynthesis of aptamer and protein microarrays in a microfluidic format; these microarrays can be immediately used for real-time SPRI bioaffinity sensing measurements.

Silica nanowire arrays for diffraction-based bioaffinity sensing.

Arrays of electrodeposited silica nanowires (SiO2 NWs) have been fabricated over large areas (cm(2)) on fluoropolymer thin films attached to glass substrates by a combination of photolithography and electrochemically triggered sol-gel nanoscale deposition. Optical and scanning electron microscopy (SEM) measurements revealed that the SiO2 NW arrays had an average spacing of ten micrometers and an average width of 700 nm with a significant grain structure that was a result of the sol-gel deposition process. The optical diffraction properties at 633 nm of the SiO2 NW arrays were characterized when placed in contact with solutions by using a prism-coupled total internal reflection geometry; quantification of changes in these diffraction properties was applied in various sensing applications. Bulk refractive index sensing by using the SiO2 NW grating was demonstrated with a sensitivity of 1.30×10(-5) RIU. Toposelectively chemically modified SiO2 NW arrays were used for diffraction biosensing measurements of surface binding events, such as the electrostatic adsorption of gold nanoparticles and the bioaffinity adsorption of streptavidin onto a biotin monolayer. Finally, the application of the SiO2 NW arrays for practical medical-diagnostic applications was demonstrated by monitoring the diffraction of SiO2 NW arrays functionalized with a single-stranded (ss)DNA aptamer to detect human α-thrombin from solutions at sub-pathologic nanomolar concentrations.

Shrink-induced silica multiscale structures for enhanced fluorescence from DNA microarrays.

We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30-45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays.

Fabrication of broadband antireflective plasmonic gold nanocone arrays on flexible polymer films.

Flexible broadband antireflective and light-absorbing nanostructured gold thin films are fabricated by gold vapor deposition onto Teflon films modified with nanocone arrays. The nanostructures are created by the oxygen plasma etching of polystyrene bead monolayers on Teflon surfaces. The periodicity and height of the nanocone arrays are controlled by the bead diameter and the overall etching time. The gold nanocone arrays exhibit a reflectivity of less than 1% over a wide spectral range (450-900 nm) and a wide range of incident angles (0-70°); this unique optical response is attributed to a combination of diffractive scattering loss and localized plasmonic absorption. In addition to nanocones, periodic nanostructures of nanocups, nanopyramids, and nanocavities can be created by the plasma etching of colloidal bilayers. This fabrication method can be used to create flexible nanocone-structured gold thin films over large surface areas (cm(2)) and should be rapidly incorporated into new technological applications that require wide-angle and broadband antireflective coatings.

DNAzyme footprinting: detecting protein-aptamer complexation on surfaces by blocking DNAzyme cleavage activity.

A novel method to quantitatively measure the binding of proteins to single-stranded DNA (ssDNA) aptamers that employs the inhibition of the DNAzyme hydrolysis of aptamer monolayers is described. A 28-base DNAzyme was designed to specifically bind to and cleave a 29-base ssDNA sequence that can fold into a G-quartet aptamer and bind the protein thrombin. The binding strength of the DNAzyme to the aptamer sequence was designed to be less than the binding strength of the thrombin to the aptamer (ΔG° = -43.1 and -51.8 kJ/mol, respectively). Formation of the thrombin-aptamer complex was found to block DNAzyme cleavage activity both in solution and in an ssDNA aptamer monolayer. We denote this method for detecting protein-aptamer complexation as "DNAzyme footprinting" in analogy to the process of DNase footprinting for the detection of protein-DNA interactions. By attaching a 40-base reporter sequence to the ssDNA aptamer monolayer, the detection of any protein-aptamer complexes remaining on the surface after DNAzyme activity can be greatly enhanced (down to one thrombin-aptamer complex per 10,000 ssDNA molecules corresponding to 100 fM thrombin in solution) by a subsequent surface RNA transcription amplification reaction followed by RNA detection with nanoparticle-enhanced SPR imaging. In addition to RNA transcription, DNAzyme footprinting can be coupled to a wide variety of other nucleic acid surface amplification schemes and thus is a powerful new route for the enzymatically amplified detection of proteins via protein-aptamer complex formation.

Electrodeposition of polydopamine thin films for DNA patterning and microarrays.

The controlled electrodeposition of functional polydopamine (PDA) thin films from aqueous dopamine solutions is demonstrated with a combination of electrochemistry, atomic force microscopy (AFM), and surface plasmon resonance (SPR) measurements. PDA micropatterns are then fabricated by electrodeposition on micrometer length scale gold electrodes and used for attaching amino-modified single-stranded DNA (ssDNA). After hybridization with fluorescently labeled ssDNA, the fluorescence microscopy characterization reveals that: (i) PDA can be toposelectively deposited at the microscale and (ii) electrochemically deposited PDA can be functionalized with amino-terminated ssDNA using the same chemistry as that for spontaneously deposited PDA. Finally, the application of electrodeposited PDA thin films to fabricate ssDNA microarrays is reported using SPR imaging (SPRI) measurements for the detection of DNA and DNA-modified gold nanoparticles.

Fabrication of DNA microarrays on polydopamine-modified gold thin films for SPR imaging measurements.

Polydopamine (PDA) films were fabricated on thin film gold substrates in a single-step polymerization-deposition process from dopamine solutions and then employed in the construction of robust DNA microarrays for the ultrasensitive detection of biomolecules with nanoparticle-enhanced surface plasmon resonance (SPR) imaging. PDA multilayers with thicknesses varying from 1 to 5 nm were characterized with a combination of scanning angle SPR and AFM experiments, and 1.3 ± 0.2 nm PDA multilayers were chosen as an optimal thickness for the SPR imaging measurements. DNA microarrays were then fabricated by the reaction of amine-functionalized single-stranded DNA (ssDNA) oligonucleotides with PDA-modified gold thin film microarray elements, and were subsequently employed in SPR imaging measurements of DNA hybridization adsorption and protein-DNA binding. Concurrent control experiments with non-complementary ssDNA sequences demonstrated that the adhesive PDA multilayer was also able to provide good resistance to the nonspecific binding of biomolecules. Finally, a series of SPR imaging measurements of the hybridization adsorption of DNA-modified gold nanoparticles onto mixed sequence DNA microarrays were used to confirm that the use of PDA multilayer films is a simple, rapid, and versatile method for fabricating DNA microarrays for ultrasensitive nanoparticle-enhanced SPR imaging biosensing.

On-chip synthesis of protein microarrays from DNA microarrays via coupled in vitro transcription and translation for surface plasmon resonance imaging biosensor applications.

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of mRNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and antiluciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications.

Wafer-scale fabrication of nanofluidic arrays and networks using nanoimprint lithography and lithographically patterned nanowire electrodeposition gold nanowire masters.

Wafer scale (cm(2)) arrays and networks of nanochannels were created in polydimethylsiloxane (PDMS) from a surface pattern of electrodeposited gold nanowires in a master-replica process and characterized with scanning electron microscopy (SEM), atomic force microscopy (AFM), and fluorescence imaging measurements. Patterns of gold nanowires with cross-sectional dimensions as small as 50 nm in height and 100 nm in width were prepared on silica substrates using the process of lithographically patterned nanowire electrodeposition (LPNE). These nanowire patterns were then employed as masters for the fabrication of inverse replica nanochannels in a special formulation of PDMS. SEM and AFM measurements verified a linear correlation between the widths and heights of the nanowires and nanochannels over a range of 50 to 500 nm. The PDMS replica was then oxygen plasma-bonded to a glass substrate in order to create a linear array of nanofluidic channels (up to 1 mm in length) filled with solutions of either fluorescent dye or 20 nm diameter fluorescent polymer nanoparticles. Nanochannel continuity and a 99% fill success rate was determined from the fluorescence imaging measurements, and the electrophoretic injection of both dye and nanoparticles in the nanochannel arrays was also demonstrated. Employing a double LPNE fabrication method, this master-replica process was also used to create a large two-dimensional network of crossed nanofluidic channels.

Near infrared surface plasmon resonance phase imaging and nanoparticle-enhanced surface plasmon resonance phase imaging for ultrasensitive protein and DNA biosensing with oligonucleotide and aptamer microarrays.

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.

On-chip synthesis of RNA aptamer microarrays for multiplexed protein biosensing with SPR imaging measurements.

Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.

Wide-field, surface-sensitive four-wave mixing microscopy of nanostructures.

We describe a wide-field four-wave mixing (FWM) microscope with imaging characteristics optimized for examining nanostructures. The microscope employs surface-plasmon polariton (SPP) excitation in a gold film to achieve surface-sensitive imaging conditions. The SPP surface fields boost the FWM efficiency by 2 orders of magnitude relative to the excitation efficiency of the evanescent fields at a bare glass surface. We demonstrate two excitation geometries that completely suppress the electronic FWM response of the metal film while allowing the far-field detection of FWM radiation from nanostructures at the interface. We obtained wide-field FWM images from individual carbon nanotubes and nanoclusters of neocyanine molecules at image acquisition times of 1 s, demonstrating the potential for background free, surface-enhanced FWM imaging of nanomaterials.

Ultrasensitive DNA microarray biosensing via in situ RNA transcription-based amplification and nanoparticle-enhanced SPR imaging.

DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications. The sensitivity and selectivity of multiplexed single-stranded DNA (ssDNA) surface bioaffinity sensing can be greatly enhanced when coupled to a surface enzymatic reaction. Herein we describe a novel method where the specific sequence-dependent adsorption of a target ssDNA template molecule onto an ssDNA-modified gold microarray is followed with the generation of multiple copies of ssRNA via in situ surface transcription by RNA polymerase. The RNA created on this "generator" element is then detected by specific adsorption onto a second adjacent "detector" element of ssDNA that is complementary to one end of the ssRNA transcript. SPR imaging is then used to detect the subsequent hybridization of cDNA-coated gold nanoparticles with the surface-bound RNA. This RNA transcription-based, dual element amplification method is used to detect ssDNA down to a concentration of 1 fM in a volume of 25 µL (25 zeptomoles).

Ultrasensitive microarray detection of short RNA sequences with enzymatically modified nanoparticles and surface plasmon resonance imaging measurements.

A novel multiplexed method for short RNA detection that employs an enzymatic capture reaction onto DNA-modified silica nanoparticles (SiNPs) followed by nanoparticle-enhanced surface plasmon resonance imaging (SPRI) is demonstrated. SiNPs functionalized with 5'-phosphorylated single stranded DNA (ssDNA) are used with T4 RNA ligase to capture various short 20-24 base single-stranded RNA (ssRNA) oligonucleotides from a target solution. The ssRNA-modified SiNPs are collected from the target solution, specifically adsorbed onto a cDNA microarray and then detected with SPRI. The use of DNA-modified SiNPs to capture ssRNA for profiling has several advantages as compared to a planar SPRI surface bioaffinity adsorption format: (i) the target solution is exposed to a larger total surface area for the RNA ligation reaction; (ii) the SiNPs enhance the diffusion rate of the ssRNA to the surface; (iii) the SiNPs can be collected, washed, and preconcentrated prior to detection; and (iv) the ssRNA-modified SiNPs give an enhanced SPRI signal upon hybridization adsorption to the microarray. Our initial measurements demonstrate that this detection method can be used to detect multiple ssRNA sequences at concentrations as low as 100 fM in 500 µL.

Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays.

The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 µm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique: First, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8 nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six-component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.

Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements.

A four-chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 µL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The 12-element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm(2) area; 45 nm thickness), each in individually addressable 0.5 µL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA, respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (ssDNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements are also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss is proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18 000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 s in the microliter volume format.

Complex Resonant Scattering Behavior in the Surface Plasmon Resonance Imaging Microscopy of Single Gold Nanorods.

Diffraction patterns observed in surface plasmon resonance imaging (SPRI) microscopy measurements of single gold nanorods (AuNRs) exhibit a complex behavior at wavelengths near the longitudinal plasmonic resonance band. SPRI microscopy measurements at 814 nm from AuNRs in three samples with resonance extinction maxima at 670, 816, and 980 nm reveal a variety of diffraction patterns with central peaks that are either positive, negative, or biphasic. A unitless ratio parameter MR (-1 ≤ MR ≤ 1) is created to describe the distribution of diffraction patterns. A purely negative (MR = -1) central peak is observed for 30%, 57%, and 98% of the diffraction patterns in the 670, 816, and 980 nm samples, respectively. These results along with a theoretical modeling of the diffraction patterns with an anisotropic complex scattering coefficient suggests that this behavior only occurs for AuNRs when the laser wavelength used in SPRI experiments is shorter than the AuNR plasmonic resonance maxima, that is, in the anomalous dispersion region.

Attomole detection of mesophilic DNA polymerase products by nanoparticle-enhanced surface plasmon resonance imaging on glassified gold surfaces.

DNA microarrays are invaluable tools for biosensing applications such as diagnostic detection of DNA and analysis of gene expression. Surface plasmon resonance imaging can detect unlabeled oligonucleotide targets adsorbed to the array elements. The variety of biosensing applications can be expanded by enzymatic manipulation of DNA microarray elements, and the sensitivity of detection can be enhanced with the use of oligonucleotide immobilized onto a gold nanoparticle surface. We describe a novel method that couples a template-directed polymerase extension of a surface array element with nanoparticle-enhanced detection of the reaction product. Using this technique, it is possible to see as little as 10-100 amol of polymerase product, representing as little as 0.25% of a monolayer. This sensitivity would allow for the detection of a specific DNA target that is present in low amounts in a sample and with partially unknown sequence. One application of this method would be to identify the presence of the aberrantly recombined DNA sequences, such as those found in the fragile sites of chromosomes.