Antje J. Baeumner

Miniaturized isothermal nucleic acid amplification, a review.

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

RNA biosensor for the rapid detection of viable Escherichia coli in drinking water.

A highly sensitive and specific RNA biosensor was developed for the rapid detection of viable Escherichia coli as an indicator organism in water. The biosensor is coupled with protocols developed earlier for the extraction and amplification of mRNA molecules from E. coli [Anal. Biochem. 303 (2002) 186]. However, in contrast to earlier detection methods, the biosensor allows the rapid detection and quantification of E. coli mRNA in only 15-20 min. In addition, the biosensor is portable, inexpensive and very easy to use, which makes it an ideal detection system for field applications. Viable E. coli are identified and quantified via a 200 nt-long target sequence from mRNA (clpB) coding for a heat shock protein. For sample preparation, a heat shock is applied to the cells prior to disruption. Then, mRNA is extracted, purified and finally amplified using the isothermal amplification technique Nucleic acid sequence-based amplification (NASBA). The amplified RNA is then quantified with the biosensor. The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The various biosensor components such as DNA probe sequences and concentration, buffers, incubation times have been optimized, and using a synthetic target sequence, a detection limit of 5 fmol per sample was determined. An excellent correlation to a much more elaborate and expensive laboratory based detection system was demonstrated, which can detect as few as 40 E. coli cfu/ml. Finally, the assay was tested regarding its specificity; no false positive signals were obtained from other microorganisms or from nonviable E. coli cells.

Development of a microfluidic biosensor module for pathogen detection

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.

Liposomes in analyses

The use of liposomes as analytical and bioanalytical reagents has been shown to be successful of in a variety of different applications that will be reviewed here. Due to their high surface area, large internal volume, and ability to conjugate bilayer lipids with a variety of biorecognition elements liposomes have been used in homogenous and heterogeneous assays, providing signal amplification both as intact or lysed vesicles. This review covers the discussion of their application in recent liposome-based immunoassay publications and includes the growing number of other non-immunoassay applications as an evidence of their immense versatility. In this article, a general background about liposomes is given first that extends past the use of liposomes as analytical tools. The main discussion is then divided by the manner in which liposomes are utilized as signaling reagents for the assays. Where available, the detection limits for common analytes that have been assayed using multiple liposome-based detection systems are presented. The advantages of using liposomes in terms of sensitivity versus other techniques are also discussed.

Analysis of liposomes

Liposomes are highly versatile structures for research, therapeutic, and analytical applications. In order to assess the quality of liposomes and obtain quantitative measures that allow comparison between different batches of liposomes, various parameters should be monitored. For liposomes used in analytical and bioanalytical applications, the main characteristics include the average diameter and degree of size polydispersity; encapsulation efficiency; the ratio of phospholipids to encapsulant concentration; lamellarity determination. A detailed description of todays most commonly used methods and of novel techniques for the quantification of these aspects is presented in this report citing 182 references. Their advantages and limitations are discussed where appropriate in order to provide the reader with an understanding of the current state of the art assessment of liposome quality.

Electrochemical microfluidic biosensor for the detection of nucleic acid sequences

A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly(dimethylsiloxane) (PDMS). The assembly was packaged into a Plexiglas housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple-potassium hexacyanoferrate (ii)/hexacyanoferrate (iii) (ferri/ferrocyanide). A combined concentration of 0.5 microM of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15-20 min of pre-incubation and required only a simple potentiostat for signal recording and interpretation.

PMMA biosensor for nucleic acids with integrated mixer and electrochemical detection.

This paper discusses the design, microfabrication and use of an electrochemical biosensor based on a polymer substrate for cost effectiveness and disposability. As model analyte, amplified hsp70 mRNA from Cryptosporidium parvum was chosen. Microfluidic channels were fabricated in poly(methyl methacrylate) (PMMA) using hot embossing with a copper master. The electrochemical transducer, an interdigitated ultramicroelectrode array (IDUA) was also realized directly on the PMMA surface. First, the unstructured PMMA surface was UV functionalized. An 8 min UV treatment resulted in a carboxylic acid density of approximately 8 nmol/cm(2) on the PMMA surface. The surface carboxylic acid groups were then conjugated to cystamine using water-soluble carbodiimide chemistry. Gold (200 nm) was then evaporated onto the thiol-functionalized surface. Using standard photolithography techniques, the IDUA containing 10 microm wide electrodes with 5 microm gaps was then formed followed by a gold etch. The PMMA surface containing the microchannel was subsequently bonded to the PMMA surface containing the IDUA using UV-assisted thermal bonding. The additional UV treatment also served to decrease the water contact angle of the surface from 62.5 degrees +/-0.7 degrees to 48.4 degrees +/-0.2 degrees thus, aiding with the capillary flow in the device. The hsp70 mRNA was isolated from C. parvum oocysts and amplified using nucleic acid sequence-based amplification (NASBA). The amplicon was detected in a sandwich hybridization assay with capture probe-coated superparamagnetic beads and reporter probe-tagged liposomes. The liposomes entrapped potassium ferro/ferrihexacyanide to enable amperometric quantification of the amplicon on the IDUA. Amplified mRNA from only 1 oocyst was detectable with this PMMA biosensor. The final detection device measured approximately 10 mm x 40 mm x 3 mm and contained two detection channels for dual analyses.

Biosensors for the detection of waterborne pathogens.

Waterborne bacterial, viral and parasitic pathogens are a global health concern and their rapid and specific detection in contaminated potable water is of utmost importance. Biosensors using a variety of biorecognition molecules and transduction methodologies have been reported, and have the potential to enable highly sensitive detection of the analyte of interest in a short time with high specificity. However, there are several obstacles to the detection of waterborne pathogens—they tend to be present at very low concentrations in the environment and environmental samples contain numerous inhibitors of enzymatic reactions and interfering organisms and particulates. Here we present a review of the current state of biosensor technology with regard to the improvements needed over standard detection methods and the challenges presented by real environmental samples. Further, we identify future areas of focus necessary to realize novel detection devices capable of supplanting the gold standards of today.

On-chip spectrophotometry for bioanalysis using microring resonators

We measure optical absorption in color-producing enzymatic reactions for biochemical analysis with a microscale optofluidic device. Cavity-enhanced laser spectrophotometry is performed on analytes within a microfluidic channel at visible wavelengths with silicon nitride microring resonators of 100 µm radius and quality factor of ~180,000. The resonator transmission spectrum is analyzed to determine optical absorption with a detection limit of 0.12 cm−1. The device can be used to detect the activity of individual enzymes in a few minutes within a 100 fL sensing volume. The high sensitivity, small footprint, and low analyte consumption make absorption-based microring resonators attractive for lab-on-a-chip applications.

Microfluidic Isolation of Nucleic Acids

The detection of nucleic acids (NAs) within micro total analysis systems (μTASs) for point-of-care use is a rapidly developing research area. The efficient isolation of NAs from a raw sample is crucial for these systems to be maximally effective. The use of microfluidics assists in reducing sample sizes and reagent consumption, increases speed, avoids contamination, and enables automation. Through miniaturization into microchips, new techniques have been realized that would be unfavorable and inconvenient to use on a macroscopic scale, but provide an excellent platform for the purification of NAs on a microscopic scale. This Review considers the complexities of NA isolation with miniaturized and microfluidic devices, as well as the considerations when choosing a technique for microfluidic NA isolation, along with their advantages, disadvantages, and potential applications. The techniques presented include using silica-based surfaces, functionalized paramagnetic beads, oligonucleotide-modified polymer surfaces, pH-dependent charged surfaces, Al2O3 membranes, and liquid-phase isolation. This Review provides a basis to develop the chemistry to improve NA isolation and move it toward achieving 100% efficiencies.