ABSTRACT
We present an integrated microelectronic device for amplification and label-free detection of nucleic acids. Amplification by polymerase chain reaction (PCR) is achieved with on-chip metal resistive heaters, temperature sensors, and microfluidic valves. We demonstrate a rapid thermocycling with rates of up to 50 degrees C s(-1) and a PCR product yield equivalent to that of a bench-top system. Amplicons within the PCR product are detected by their intrinsic charge with a silicon field-effect sensor. Similar to existing optical approaches with intercalators such as SYBR Green, our sensing approach can directly detect standard double-stranded PCR product, while in contrast, our sensor does not require labeling reagents. By combining amplification and detection on the same device, we show that the presence or absence of a particular DNA sequence can be determined by converting the analog surface potential output of the field-effect sensor to a simple digital true/false readout.
Subject(s)
Electronics , Nucleic Acids/chemistry , Polymerase Chain Reaction/instrumentation , Systems Integration , Base Sequence , DNA Primers , Nucleic Acids/analysis , Sensitivity and SpecificityABSTRACT
Heparin is a highly sulfated glycosaminoglycan that is used as an important clinical anticoagulant. Monitoring and control of the heparin level in a patient's blood during and after surgery is essential, but current clinical methods are limited to indirect and off-line assays. We have developed a silicon field-effect sensor for direct detection of heparin by its intrinsic negative charge. The sensor consists of a simple microfabricated electrolyte-insulator-silicon structure encapsulated within microfluidic channels. As heparin-specific surface probes the clinical heparin antagonist protamine or the physiological partner antithrombin III were used. The dose-response curves in 10% PBS revealed a detection limit of 0.001 units/ml, which is orders of magnitude lower than clinically relevant concentrations. We also detected heparin-based drugs such as the low-molecular-weight heparin enoxaparin (Lovenox) and the synthetic pentasaccharide heparin analog fondaparinux (Arixtra), which cannot be monitored by the existing near-patient clinical methods. We demonstrated the specificity of the antithrombin III functionalized sensor for the physiologically active pentasaccharide sequence. As a validation, we showed correlation of our measurements to those from a colorimetric assay for heparin-mediated anti-Xa activity. These results demonstrate that silicon field-effect sensors could be used in the clinic for routine monitoring and maintenance of therapeutic levels of heparin and heparin-based drugs and in the laboratory for quantitation of total amount and specific epitopes of heparin and other glycosaminoglycans.
Subject(s)
Anticoagulants/chemistry , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Drug Monitoring , Heparin, Low-Molecular-Weight/chemistry , Heparin/chemistry , Silicon/chemistry , Adsorption , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Antithrombin III/chemistry , Antithrombin III/physiology , Carbohydrate Sequence , Colorimetry , Dose-Response Relationship, Drug , Drug Monitoring/methods , Enoxaparin/chemistry , Enoxaparin/pharmacology , Enoxaparin/therapeutic use , Factor Xa/analysis , Fondaparinux , Forecasting , Heparin/pharmacology , Heparin/therapeutic use , Heparin, Low-Molecular-Weight/pharmacology , Heparin, Low-Molecular-Weight/therapeutic use , Humans , Kinetics , Microfluidics , Polysaccharides/chemistry , Polysaccharides/pharmacology , Polysaccharides/therapeutic use , Protamines/antagonists & inhibitors , Protamines/metabolism , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We present a robust and simple method for direct, label-free PCR product quantification using an integrated microelectronic sensor. The field-effect sensor can sequentially detect the intrinsic charge of multiple unprocessed PCR products and does not require sample processing or additional reagents in the PCR mixture. The sensor measures nucleic acid concentration in the PCR relevant range and specifically detects the PCR products over reagents such as Taq polymerase and nucleotide monomers. The sensor can monitor the product concentration at various stages of PCR and can generate a readout that resembles that of a real-time fluorescent measurement using an intercalating dye but without its potential inhibition artifacts. The device is mass-produced using standard semiconductor processes, can be reused for months, and integrates all sensing components directly on-chip. As such, our approach establishes a foundation for the direct integration of PCR-based in vitro biotechnologies with microelectronics.