Use of porous membranes modified with polyelectrolyte multilayers as substrates for protein arrays with low nonspecific adsorption

Coating of substrates with polyelectrolyte multilayers terminated with poly(acrylic acid) (PAA) followed by activation of the free -COOH groups of PAA provides a surface that readily reacts with amine groups to allow covalent immobilization of antibodies. The use of this procedure to prepare arrays of antibodies in porous alumina supports facilitates construction of a flow-through system for analysis of fluorescently labeled antigens. Detection limits in the analysis of Cy5-labeled IgG are 0.02 ng/mL because of the high surface area of the alumina membrane, and the minimal diameter of the substrate pores results in binding limited by kinetics, not mass transport. Moreover, PAA-terminated films resist nonspecific protein adsorption, so blocking of antibody arrays with bovine serum albumin is not necessary. These microarrays are capable of effective analysis in 10% fetal bovine serum.     

Nanoliter dispensing method by degassed poly(dimethylsiloxane) microchannels and its application in protein crystallization

This paper describes a method of dispensing a nanoliter volume of liquid into arrays of microwells through degassed poly(dimethylsiloxane) (PDMS) microchannels. In this method, the PDMS microchannels were reversibly bound to arrays of microwells. The PDMS elastomer was predegassed and served as an internal vacuum pumping source. Various aqueous solutions were infused into arrays of microwells through the reversibly sealed PDMS microchannels. Microwells fabricated in PDMS, poly(methyl methacrylate) (PMMA), and glass were all compatible with this dispensing method. By removing the PDMS microchannels, arrays of droplets confined in the microwells were obtained. Multiplex reaction and screening at the nanoliter scale were carried out by binding two such arrays of microwells to form microchambers. We applied this method to screening the crystallization conditions of four known proteins. Long-term incubation of over 2 months was achieved by employing glass microwells. An unknown protein was then crystallized using the screening method in microwells. The crystals with sufficient size were harvested from the reversibly bound microwells. X-ray diffraction with a resolution of 3.1 Angstrom was obtained.     

Optimal conditions for protein array deposition using continuous flow

Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.     

Patterning Nanostructured,Synthetic, Polymeric Receptors by Simultaneous Projection Photolithography,Nanomolding, and Molecular Imprinting

Microscope projection photolithography is combined with nanomolding and molecular imprinting for the fast microfabrication of molecularly imprinted polymer (MIP) arrays in the form of micrometric islands of nanofilaments. Dot diameters from 70–90 μm are easily obtained using a 10× objective and a photomask carrying the desired pattern. The dots are composed of parallel nanofilaments of a high aspect ratio, 150 nm in diameter and several micrometers in length, which are obtained through a nanomolding procedure on porous alumina. The arrays are molecularly imprinted with the small molecule fluorescein or with the protein myoglobin. The fluorescein MIP arrays are able to specifically recognize their target, as demonstrated by fluorescence microscopy. A four‐fold increase in binding capacity and imprinting factor (IF = 13) is obtained compared to non‐nanostructured porous dots. Imprinting of the nanofilament arrays with the protein myoglobin as the template is also possible and allows for a high imprinting factor of 4.3. Such nanostructured microarrays of synthetic receptors obtained by projection photolithography have great potential in biosensor and biochip development.     

Development of surface plasmon resonance mass spectrometry array platform

Protein microarrays are the format of choice for high-throughput, high-content protein interaction analysis. In most of the array formats, reporter molecules are used in multistep detection of the protein interactions. Among the few existing label-free detection approaches, surface plasmon resonance (SPR) and mass spectrometry (MS) stand out as most promising for utilization in protein microarrays, albeit both have been used only sporadically for high-content protein arrays. Shown here for the first time is the combination of SPR and MS detection on a single high-content protein microarray. Antibodies to five human plasma proteins were arrayed in a 10 x 10 spot arrangement on a chemically activated gold-coated glass chip. Binding of proteins to their corresponding antibodies was monitored via SPR imaging across the entire surface of the chip. Following protein affinity retrieval, the chip was overlaid with MALDI matrix and MS analyzed, producing protein-specific mass spectra from distinct spots on the array. The SPR-MS dual detection is well suited for high-content protein microarrays and comprehensive protein analysis-from quantitative assessment of the protein concentration to detection of structural protein variants arising from genetic variations and postexpression processing.     

Porous SiO2 interferometric biosensor for quantitative determination of protein interactions: binding of protein A to immunoglobulins derived from different species

Determination of kinetic and thermodynamic protein binding constants using interferometry from a porous Si Fabry-Perot layer is presented. A protein A capture probe is adsorbed within the pores of an oxidized porous Si matrix, and binding of immunoglobulin G (IgG) antibodies derived from different species is investigated. The relative protein A/IgG binding affinity is human > rabbit > goat, in agreement with literature values. The equilibrium binding constant (Ka) for human IgG binding to surface-immobilized protein A is determined to be (3.0 +/- 0.5) x 107 M-1 using an equilibrium Langmuir model. Kinetic rate constants are calculated to be kd = (2.1 +/- 0.2) x 10-4 s-1 and ka = (1.2 +/- 0.4) x 104 M-1 s-1 using nonlinear least-squares analysis, yielding an equilibrium binding constant of Ka = (5.5 +/- 1.5) x 107 M-1. Both steady-state and time-dependent measurements yield equilibrium binding constants that are consistent with literature values. Kinetic rate constants determined through nonlinear least-squares analysis are also in agreement with protein A/IgG binding on a surface. Dosing with a high concentration of analyte leads to deviations from ideal binding behavior, interpreted in terms of restricted analyte diffusion within the porous SiO2 matrix. It is shown that the diffusion limitations can be minimized if the kinetic measurements are performed at low analyte concentrations or under conditions in which the protein A capture probe is not saturated with analyte. Potential limitations of the use of porous SiO2 interferometers for quantitative determination of protein binding constants are discussed.     

High-throughput immunophenotyping by surface plasmon resonance imaging

Cell binding assays on antibody arrays permit the rapid immunophenotyping of living cells. The throughput of the analysis, however, is still limited due to our inability to perform parallel and quantitative detection of cells captured on the array. To address this limitation, we employed here an imaging technique based on surface plasmon resonance (SPR). SPR has been frequently used to monitor capture of proteins on antibody microarrays, while few cases were reported for capture of cells. Antibody arrays were prepared through the photopatterning of an alkanethiol monolayer on a gold-evaporated glass plate and the subsequent immobilization of various antibodies onto 4-9 separate spots created by photopatterning. A glass slip was mounted onto the array with a thin spacer to construct a parallel-plate chamber. Leukemia cells were injected into the chamber to conduct a binding assay, while refractive index changes at the vicinity of the array surface were monitored by SPR imaging. We observed that SPR signals were intensified on specific antibody spots but not on nonspecific spots. Confocal laser scanning microscopy revealed that the observed SPR signals were attributed to cell deformations caused by multivalent interactions with immobilized antibody, which effectively elevated the refractive index of a medium phase within an evanescent field. This effect could be suitably utilized to monitor quantitatively cell binding to multiple spots from a heterogeneous cell population.     

A microarray immunoassay for simultaneous detection of proteins and bacteria

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).     

High-speed fluorescence detection of explosives-like vapors

In this paper, we report on the preparation of novel cross-reactive optical microsensors for high-speed detection of low-level explosives and explosives-like vapors. Porous silica microspheres with an incorporated environmentally sensitive fluorescent dye are employed in high-density sensor arrays to monitor fluorescence changes during nitroaromatic compound (NAC) vapor exposure. The porous silica-based sensor materials have good adsorption characteristics, high surface areas, and surface functionality to help maximize analyte-dye interactions. These interactions occur immediately upon vapor exposure, i.e., in less than 200 ms and are monitored with a high-speed charge-coupled device camera to produce characteristic and reproducible vapor response profiles for individual sensors within an array. Employing thousands of identical microsensors permits sensor responses to be combined, which significantly reduces sensor noise and enhances detection limits. Normalized response profiles for 1,3-dinitrobenzene (1,3-DNB) are independent of analyte concentration, analyte exposure time, or sensor age for an array of one sensor type. Explosives-like NACs such as 2,4-dinitrotoluene and DNB are detected at low part-per-billion levels in seconds. Sensor-analyte profiles of some sensor types are more sensitive to low-level NAC vapor even when in a higher organic vapor background. We show that single-element arrays permit the detection of low-level nitroaromatic compound vapors because of sensor-to-sensor reproducibility and signal averaging.     

Magnetic assembly of high-density DNA arrays for genomic analyses

A method for rapidly assembling high-density DNA arrays with near-perfect order is described. Photolithography is used to generate a wafer-scale array of microwells in a layer of photoresist on a chemically functionalized glass coverslip. The array is enclosed within a microfluidic device, and a suspension of superparamagnetic microbeads conjugated to DNA molecules is introduced into the chamber. A permanent magnet is used to direct the rapid assembly of the beads into the wells, with each well containing a single bead. These beads are immobilized on the glass surface via affinity binding, and excess beads can be recycled or washed away. Nonspecifically bound beads are removed by dissolving the photoresist. The result is a high-density array of beads with virtually no background. This method can be used to produce protein arrays for chip-based assays and DNA arrays for genotyping or genome sequencing.     

Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, alpha-1-fetoprotein and beta2-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.     

Label-free reading of microarray-based immunoassays with surface plasmon resonance imaging

A simple method is presented for patterning of protein antigens at a gold surface for use in surface plasmon resonance (SPR) imaging experiments. Microfluidic devices fabricated from poly(dimethylsiloxane) were used to flow various fluids over a gold substrate in spatially defined channels. This technique was used to pattern the surface chemistry of the gold as well as to adsorb antigens from solution to the modified substrates. The resulting antigen arrays were probed with complementary antibodies in order to demonstrate the effectiveness of the patterning for antibody capture experiments. SPR imaging was used to aid in the optimization of array fabrication and to observe the interactions of unlabeled antibodies with these microarrays. This work presents a means of fabricating microarrays with controlled surface density of antigens. SPR imaging provides both quantitative and qualitative evaluation of antibody binding in a label free format.     

3D protein microarrays: performing multiplex immunoassays on a single chip

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.     

Large-scale patterning of gold nanopillars in a porous anodic alumina template by replicating gold structures on a titanium barrier

Gold nanopillars are grown in patterns inside a porous anodic alumina template. On selected positions, defined by a gold "seed" pattern, gold is electroplated into the pores, while a barrier layer underneath the porous template blocks the deposition on the rest of the surface. Large-scale arrays of free-standing nanopillar islands are obtained after selective etching of the alumina template.     

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

Removal of Nonspecifically Bound Proteins on Microarrays Using Surface Acoustic Waves

Nonspecific binding of proteins is an ongoing problem that dramatically reduces the sensitivity and selectivity of biosensors. We demonstrate that ultrasonic waves generated by surface acoustic wave (SAW) devices remove nonspecifically bound proteins from the sensing and nonsensing regions of the microarrays. We demonstrate our approach for controllably and nondestructively cleaning the microarray interface. In this work, SAWs were generated using 128 YX lithium niobate, chosen for its high coupling coefficient and efficient power transfer to mechanical motion. These waves propagating along the surface were coupled into specifically bound and nonspecifically bound proteins on a patterned surface of 40 mum feature size. Fluorescence intensity was used to quantify cleaning efficacy of the microarrays. Our results have shown that excess protein layers and aggregates are removed leaving highly uniform films as evidenced by fluorescence intensity profiles. Selected antigen-receptor interactions remained bound during the acoustic cleaning process when subjected to 11.25 mW of power and retained their efficacy for subsequent antigen capture. Results demonstrate near-complete fluorescence signal recovery for both the sensing and nonsensing regions of the microarrays. Of significance is that our approach can be integrated into existing array technologies where sensing and nonsensing regions are extensively fouled. We believe that this technology will be pivotal in the development and advancement of microsensors and their biological applications.     

Electrostatic protein immobilization using charged polyacrylamide gels and cationic detergent microfluidic Western blotting

We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The "pan-analyte" capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sj?gren's Syndrome, an autoimmune disease.     

Competitive immunoassay for microliter protein samples with magnetic beads and near-infrared fluorescence detection

A competitive immunoassay with near-infrared (NIR) fluorescence detection to analyze microliter biological samples with an amol limit of detection (LOD) is described. An important feature about this technique is that the immunoreaction and fluorescence detection are separated into two distinct steps, allowing for independent optimization. In the immunoreaction step, NIR fluorescence-labeled antigen (Ag) competes with the unlabeled analyte (Ag) for antibodies (Ab) immobilized on the surface of paramagnetic beads. A magnet is then used to separate the bound antigen from the free in the supernatant. As the amount of Ag in the sample increases, there is less binding between Ag and immobilized Ab; therefore, the amount of Ag in the supernatant is proportionally related to the amount of Ag in the sample. In the fluorescence detection step, aliquots of the supernatant are concentrated onto a protein binding membrane by a capillary blotting technique with an optimized 33 nL/min flow rate. The fluorescence of the blotted spots is detected with a NIR sensitive photon counting system that is optimized to an instrumental LOD of 30 000 fluorophore molecules. This competitive assay demonstrates a sample LOD of 400 pg/mL of unlabeled rabbit immunoglobulin G spiked into bovine serum. This design features low sample volumes and reagent consumption.     

Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor

The identification of binding partners of proteins by mass spectrometry following specific capture on a biosensor surface is a promising tool for proteomics research and the identification and characterization of protein-protein interactions. Previous approaches include the direct ionization of analyte from the biosensor chip on a matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS) apparatus and the on-chip digestion followed by elution, chromatographic concentration of the fragments, and electrospray mass spectrometry. In the present paper, using the small-volume microfluidic sample manipulation technique with oscillatory flow reported recently (Abrantes et al. Anal. Chem. 2001, 73, 2828-2835), analyte is shown to be eluted from the sensor surface into a small volume of buffer that promotes dissociation from the capture surface and delivery to the mass spectrometer. Both the incubation of the sensor surface with the sample and the recovery of analyte can be achieved with a few microliters and conducted until steady-state is attained. Because the procedure is non-destructive for the sensor surface, multiple cycles of capture and elution allow the transfer and concentration of analyte into the elution buffer. The eluted analyte can be studied directly by MALDI-TOFMS, or subjected to proteolytic digestion for protein identification. Transfer into the elution buffer and MALDI-TOFMS detection was achieved from 5 microL of starting samples containing <50 fmol of analyte. Examples are presented for the specific detection and recovery of a protein from a complex mixture of cytosolic proteins.     

Enhancement of Raman signals with silver-coated tips

Silver coated SiN and SiO(2) tips have been fabricated for use with a bottom-illumination tip-enhanced Raman spectroscopy (TERS) setup with a 488 nm laser excitation. SiN tips with 50-60 nm of deposited Ag give the best TERS enhancements for brilliant cresyl blue test analyte spin-coated on a glass slide. Ag nanoparticles on SiN or SiO(2) rather than Si tips are better for TERS because of the proximity of the wavelengths of their surface plasmon resonance to 488 nm. Adjustments of tilt angle of the metallized tip with respect to the surface plane is shown to considerably raise the intensities of the TERS signals, even from tips that initially appear to be rather non-enhancing. This work helps to enable the more frequent use of the 488 nm laser for nanoscale chemical analysis with both TERS and fluorescence imaging in the same setup.