David R. Walt

Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as ∼10–20 enzyme-labeled complexes in 100 μl of sample (∼10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays

We have developed a randomly ordered fiber-optic gene array for rapid, parallel detection of unlabeled DNA targets with surface immobilized molecular beacons (MB) that undergo a conformational change accompanied by a fluorescence change in the presence of a complementary DNA target. Microarrays are prepared by randomly distributing MB-functionalized 3-μm diameter microspheres in an array of wells etched in a 500-μm diameter optical imaging fiber. Using several MBs, each designed to recognize a different target, we demonstrate the selective detection of genomic cystic fibrosis related targets. Positional registration and fluorescence response monitoring of the microspheres was performed using an optical encoding scheme and an imaging fluorescence microscope system.

The chemistry of enzyme and protein immobilization with glutaraldehyde

Abstract Immobilization of proteins to solid matrices has been performed for the last thirty years and has provided numerous examples of successful preparations with use in enzyme reactors, sensor preparation and immunodiagnostics. Among the arsenal of coupling reagents and procedures, glutaraldehyde plays a critical role due to its reliability and ease of use. It displays a complex chemistry that is transparent to most practitioners of immobilization. In this article we detail the structure and reactivity of glutaraldehyde protein immobilization.

Fluorescence-based nucleic acid detection and microarrays

Abstract Recent analytical innovations for nucleic acid detection have revolutionized the biological sciences. Single nucleic acid sequence detection methods have been expanded to incorporate multiplexed detection strategies. A variety of nucleic acid detection formats are now available that can address high throughput genomic interrogation. Many of these parallel detection platforms or arrays, employ fluorescence as the signaling method. Fluorescence-based assays offer many advantages, including increased sensitivity, safety and multiplexing capabilities, as well as the ability to measure multiple fluorescence properties. Multiplexed microarray platforms provide parallel detection capabilities capable of measuring thousands of simultaneous responses. This review will discuss both single target detection and microarray applications with a focus on gene expression and pathogenic microorganism (PM) detection.

Current trends in `artificial-nose' technology

Basic principles derived from biological olfaction, such as combining semiselective sensor arrays with pattern recognition, have been used to develop instrumentation capable of broad-band chemical detection and quantification. Commercially available instruments are useful in areas including quality control in the food, beverage and fragrance industries, environmental monitoring, chemical-purity and -mixture analysis, and medical diagnostics. Ongoing research is aimed at the development of more-advanced instruments that are smaller, cheaper, faster and more stable and reliable. These second-generation instruments are likely to find an increasing number of applications, including the on-line monitoring of fermentation and other bioprocesses.

Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering

Rapid progress in identifying disease biomarkers has increased the importance of creating high-performance detection technologies. Over the last decade, the design of many detection platforms has focused on either the nano or micro length scale. Here, we review recent strategies that combine nano- and microscale materials and devices to produce large improvements in detection sensitivity, speed and accuracy, allowing previously undetectable biomarkers to be identified in clinical samples. Microsensors that incorporate nanoscale features can now rapidly detect disease-related nucleic acids expressed in patient samples. New microdevices that separate large clinical samples into nanocompartments allow precise quantitation of analytes, and microfluidic systems that utilize nanoscale binding events can detect rare cancer cells in the bloodstream more accurately than before. These advances will lead to faster and more reliable clinical diagnostic devices.

A fiber-optic sensor for CO2 measurement

A fiber-optic sensor has been prepared which responds to carbon dioxide at physiologically significant concentrations. It is based on pH modulation by dissolved carbon dioxide in a sensing layer of fluorescent dye. By use of a previously developed methodology by which the sensing chemistry is bonded directly to the glass fiber tip, the miniature size of the sensor is preserved. This method involves consecutive applications of solution polymers to the fiber tip rather than mechanical attachment of sensor reagents. Preparations of polymer-immobilized dyes and polymer membranes are described.

Multiplexed Sandwich Immunoassays using Electrochemiluminescence Imaging Resolved at the Single Bead Level

A new class of bead-based microarray that uses electrogenerated chemiluminescence (ECL) as a readout mechanism to detect multiple antigens simultaneously is presented. This platform demonstrates the possibility of performing highly multiplexed assays using ECL because all the individual sensing beads in the array are simultaneously imaged and individually resolved by ECL. Duplex and triplex assay results are demonstrated as well as a cross reactivity study.

Fluorescence-based fibre optic arrays: a universal platform for sensing

Optical fibres provide a universal sensing platform as they are easily integrated with a multitude of different sensing schemes. Such schemes enable the preparation of a multitude of sensors from relatively straightforward pH sensors, to more complex ones, including artificial olfaction sensors, high-density oligonucleotide arrays, and high-throughput cell-based arrays. Imaging fibre bundles comprised of thousands of fused optical fibres are the basis for an optically connected, individually addressable parallel sensing platform. Fibre optic imaging bundles possess miniature feature sizes (3-10 micron diameter fibres), allowing high-density sensor packing (approximately 2 x 10(7) sensors per cm2). Imaging fibre bundles transmit coherent images enabling combined imaging and sensing, relating the responses monitored by the sensor to observable physical changes. The individual fibre cores can also be selectively etched to form a high-density microwell array capable of housing complementary sized microsensors. The miniature feature sizes facilitate a faster response and more sensitive measurement capabilities. The platform is extremely versatile in its sensing design, allowing the sensing scheme to be tailored to fit the experimental design, whether for monitoring single analytes or more complex multiplexed assays. A number of sensing schemes and applications are described in this review.

Intelligent Medical Diagnostics via Molecular Logic

In this communication, we describe the integration of microarray sensor technology with logic capability for screening combinations of proteins and DNA in a biological sample. In this system, we have demonstrated the use of a single platform amenable to both protein detection and protein-DNA detection using molecular logic gates. The pattern of protein and DNA inputs results in fluorescence outputs according to a truth table for AND and INHIBIT gates, thereby demonstrating the feasibility of performing medical diagnostics using a logic gate design. One possible application of this technique would be direct screening of various medical conditions that are dependent on combinations of diagnostic markers.