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The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.

Rapid (∼10 min) measurement of very low concentration of pathogens (∼10 cells/mL) and protein (∼fg/mL) has widespread use in medical diagnostics, monitoring biothreat agents, and in a broader context as a research method. For low-level pathogen, we currently use culture enrichment methods and, thus, rapid analysis is not possible. For low protein concentration, no direct method is currently available. We report here a novel macrocantilever design whose high-order resonant mode near 1 MHz exhibits mass detection sensitivity of 10 cells/mL for cells and 100 fg/mL for protein. The sensor is 1 × 3 mm and uses a piezoelectric layer for both actuation and sensing resonance. Sample is flowed (∼1 mL/min) past the antibody-immobilized sensor, and as antigen binds to the sensor, resonance frequency decreases in proportion to antigen concentration. The sensor showed selectivity to the pathogen even though copious nonpathogenic variant was simultaneously present.Rapid and single-step label-free direct detection of proteins in flowing liquid samples at a concentration of 100 fg/mL has not been reported to date. Neither has there been any report on detecting pathogens at 10 cells per mL from samples of tens of milliliters under flow conditions. Both measurements have significant applications in medicine (for biomarkers in body fluids), 1 environmental monitoring (pathogens in drinking water), food safety (Listeria, 2 Cyrptosporidium, Giardia, 3 and Escherichia coli poisoning 4 ), and biodefense (biothreat agents). In this paper, we show designs of millimeter-sized cantilever sensors that exhibit mass change sensitivity of femtograms under liquid flow conditions and that are potentially useful in the applications mentioned above. Three types of experiments are reported to demonstrate the high detection sensitivity.Cantilever biosensors have attracted considerable interest in the past decade for label-free detection of proteins and pathogens because of their promise of very high sensitivity. 5,6 Excellent reviews have appeared that summarize progress. 7,8 Briefly, the binding of an antigenic target to an antibody-immobilized cantilever surface changes the cantilever's surface stress resulting in a deflection response. 9,10 Cantilever biosensors 11,12 have been successfully used in DNA hybridization studies, 9,13 detection of known cancer proteins, 14 environmental and foodborne pathogens, 15,16 biomarkers, 17,18 and explosives. 19 In dynamic mode, the attachment of antigen causes a resonant frequency decrease because of increase in mass. 7 Magnitude of bending deflection can be monitored by various transduction mechanisms. 7,[20][21][22] Because significant damping occurs in the dynamic mode, static deflection method is preferred when continuous measurement under liquid immersion is needed. When measurement in liquid flow condition is required, the bending mode becomes noisy and less trustworthy because of fluctuating hydrodynamic forces. It is well established 23 that for the dynamic method to provide reasonable signals, c...

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.

A sensitive, selective, sample preparation-free method for near real-time detection of microRNA in buffer and human serum is given using gold (Au)-coated dynamic piezoelectric cantilever sensors. Sensor response to thiolated DNA probe chemisorption, hsa-let-7a hybridization, labeled-DNA hybridization, and Au nanoparticle-functionalized DNA hybridization was monitored continuously in flowing liquid samples using custom flow-cells. The assay showed successful detection of target let-7a with a dynamic range spanning 6 orders of magnitude (10 fM-1 nM) with a limit of detection of less than 10 attomoles (∼4 fM). The serum background had negligible effect on sensitivity relative to the results obtained in the buffer due to reduction in nonspecific binding caused by continuous sensor vibration. Both hybridization and nonspecific binding reduction were confirmed using fluorescence-based assays to support sensor-based results. The sensor-based method demonstrated excellent selectivity for the microRNA target in comparison with similar microRNA differing by only a single nucleotide (hsa-let-7c) and random microRNA sequences. Au nanoparticle-based amplification of sensor response was investigated and led to an order of magnitude improvement in the detection limit and a 128% amplification of sensor response over the entire dynamic range. Au nanoparticle amplification was verified by scanning electron microscopy. The cantilever sensor-based microRNA assay provides competitive sensitivity with current microRNA detection methods and has the advantage of requiring no sample preparation, even when working with biological samples that contain a complex background.

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