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Improved methods are needed for routine, inexpensive monitoring of biomarkers that could facilitate earlier detection and characterization of cancer. Suspended microchannel resonators (SMRs) are highly sensitive, batch-fabricated microcantilevers with embedded microchannels that can directly quantify adsorbed mass via changes in resonant frequency. As in other label-free detection methods, biomolecular measurements in complex media such as serum are challenging due to high background signals from nonspecific binding. In this report, we demonstrate that carboxybetaine-derived polymers developed to adsorb directly onto SMR Si[O.sub.2] surfaces act as uitralow fouling and functionalizable surface coatings. Coupled with a reference microcantilever, this approach enables detection of activated leukocyte cell adhesion molecule (ALCAM), a model cancer biomarker, in undiluted serum with a limit of detection of 10 ng/mL. 10.1021/ac9027356

Cell growth comprises changes in both mass and volume--two processes that are distinct, yet coordinated through the cell cycle. Understanding this relationship requires a means for measuring each of the cell's three basic physical parameters: mass, volume, and the ratio of the two, density. The suspended microchannel resonator weighs single cells with a precision in mass of 0.1% for yeast. Here we use the suspended microchannel resonator with a Coulter counter to measure the mass, volume, and density of budding yeast cells through the cell cycle. We observe that cell density increases prior to bud formation at the G1/S transition, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator target of rapamycin, passage through START (commitment to cell division), and an intact actin cytoskeleton. Although we focus on basic cell cycle questions in yeast, our techniques are suitable for most nonadherent cells and subcellular particles to characterize cell growth in a variety of applications. biosensor | cell growth | cell size | microfluidics | Saccharomyces doi/10.1073/pnas.0901851107

We investigate the buoyant mass of bacterial cells in real time with the suspended microchannel resonator (SMR) as the population recovers from an osmotic shock. The density of the culture medium is chosen such that the bacteria initially have a positive buoyant mass which becomes negative as they recover from the hyperosmotic stress. This behavior can be used to differentiate between an antibiotic-resistant and an antibiotic-susceptible strain of the pathogenic bacteria Citrobacter rodentium, and we propose a general approach for exploiting the high precision of the SMR for rapid detection of antibiotic resistance.

Measurements of the mass and surface charge of microparticles are employed in the characterization of many types of colloidal dispersions. The suspended microchannel resonator (SMR) is capable of measuring individual particle masses with femtogram resolution. Here, we employ the high sensitivity of the SMR resonance frequency to changes in particle position, relative to the cantilever tip, to determine the electrophoretic mobility of discrete particles in an applied electric field. When a sinusoidal electric field is applied to the suspended microchannel, the transient resonance frequency shift corresponding to a particle transit can be analyzed by digital signal processing to extract both the buoyant mass and electrophoretic mobility of each particle. These parameters, together with the mean particle density, can be used to compute the size, absolute mass, and surface charge of discrete microspheres, leading to a true representation of the mean and polydispersity of these quantifies for a population. We have applied this technique to an aqueous suspension of two types of polystyrene microspheres, to differentiate them based on their absolute mass and their surface charge. The integrated measurement of electrophoretic mobility using the SMR is determined to be quantitative, based on comparison with commercial instruments, and exhibits favorable scaling properties that will ultimately enable measurements from mammalian cells.

Universal detectors that maintain high sensitivity as the detection volume is reduced to the subnanoliter scale can enhance the utility of miniaturized total analysis systems ([mu]-TAS). Here the unique scaling properties of the suspended microchannel resonator (SMR) are exploited to show universal detection in a 10 pL analysis volume with a density detection limit of ~1 [micro]g/[cm.sup.3] (10 Hz bandwidth) and a dynamic range of 6 decades. Analytes with low IN extinction coefficients such as polyethylene glycol (PEG) 8 kDa, glucose, and glycine are measured with molar detection limits of 0.66, 13.5, and 31.6 [micro]M, respectively. To demonstrate the potential for real-time monitoring, gel titration chromatography was used to separate different molecular weights of PEG as the SMR acquired a chromatogram by measuring the eluate density. This work suggests that the SMR could offer a simple and sensitive universal detector for various separation systems from liquid chromatography to capillary electrophoresis. Moreover, since the SMR is itself a microfluidic channel, it can be directly integrated into [mu]-TAS without compromising overall performance.

Author(s): Thomas P. Burg [1, 5]; Michel Godin [1, 5]; Scott M. Knudsen [1]; Wenjiang Shen [3]; Greg Carlson [3]; John S. Foster [3]; Ken Babcock [3, 4]; Scott R. [...]

There is a great need in experimental biology for tools to study interactions between biological molecules and to profile expressure levels of large numbers of proteins. This paper describes the fabrication, packaging and testing of a resonant mass sensor for the detection of biomolecules in a microfluidic format. The transducer employs a suspended microchannel as the resonating element, thereby avoiding the problems of damping and viscous drag that normally degrade the sensitivity of resonant sensors in liquid. Our device differs from a vibrating tube densitometer in that the channel is very thin, which enables the detection of molecules that bind to the channel walls; this provides a path to specificity via molecular recognition by immobilized receptors. The fabrication is based on a sacrificial polysilicon process with low-stress low-pressure chemical-vapor deposited (LPCVD) silicon nitride as the structural material, and the resonator is vacuum packaged on the wafer scale using glass frit bonding. Packaged resonators exhibit a sensitivity of 0.8 ppm/(ng * [cm.sup.2]) and a mechanical quality factor of up to 700. To the best of our knowledge, this quality factor is among the highest so far reported for resonant sensors with comparable surface mass sensitivity in liquid. Index Terms--Biomedical transducers, chemical transducers, density measurement, Q factor, mass sensor, microbalance, vacuum packaging.

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-insulatorsilicon 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. heparin sensors | label-free sensing | medical devices | enoxaparin | fondaparinux

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.

We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/ reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.

We have micromachined a mechanical sensor that uses interferometry to detect the differential and absolute deflections of two adjacent cantilevers. The overall geometry of the device allows simple fluidic delivery to each cantilever to immobilize molecules for biological and chemical detection. We show that differential sensing is 50 times less affected by ambient temperature changes than the absolute, thus enabling a more reliable differentiation between specific cantilever bending and background effects. We describe the fabrication process and show results related to the dynamic characterization of the device as a differential sensor. The root-mean-squared (rms) sensor noise in water and air is ~1 nm over the frequency range of 0.4-40 Hz. We also find that in air, the deflection resolution is limited only by the cantilever's thermomechanical noise level of 0.008 [Angstrom]/[Hz.sup.1/2] over the frequency range of 40-1000 Hz. [781] Index Terms--AFM technology, BioMEMS, biosensor, chemical-biological functionalization, diffraction, interdigitated, silicon nitride.

We report the selective and real-time detection of label-free DNA using an electronic readout. Microfabricated silicon field-effect sensors were used to directly monitor the increase in surface charge when DNA hybridizes on the sensor surface. The electrostatic immobilization of probe DNA on a positively charged poly-L-lysine layer allows hybridization at Low ionic strength where field-effect sensing is most sensitive. Nanomolar DNA concentrations can be detected within minutes, and a single base mismatch within 12-mer oligonucleotides can be distinguished by using a differential detection technique with two sensors in parallel. The sensors were fabricated by standard silicon microtechnology and show promise for future electronic DNA arrays and rapid characterization of nucleic acid samples. This approach demonstrates the most direct and simple translation of genetic information to microelectronics.

High-resolution atomic force microscopy (AFM) of soft or fragile samples requires a cantilever with a low spring constant and a sharp tip. We have developed a novel process for making such cantilevers from silicon nitride with oxidation-sharpened silicon tips. First, we made and sharpened silicon tips on a silicon wafer. Next, we deposited a thin film of silicon nitride over the tips and etched it to define nitride cantilevers and to remove it from the tips so that they protruded through the cantilevers. Finally, we etched from the back side to release the cantilevers by removing the silicon substrate. We characterized the resulting cantilevers by imaging them with a scanning electron microscope, by measuring their thermal noise spectra, and by using them to image a test sample in contact mode. A representative cantilever had a spring constant of ~0.06 N/m, and the tip had a radius of 9.2 nm and a cone angle of 36[degrees] over 3 [micro]m of tip length. These cantilevers are capable of higher resolution imaging than commercially available nitride cantilevers with oxidation-sharpened nitride tips, and they are especially useful for imaging large vertical features. [735] Index Terms--Atomic force microscopy (AFM), microfabricated cantilevers, microfabricated tips, micromachining, oxidation sharpening.

A high-resolution accelerometer with a bulk-micromachined silicon proof mass and an interferometric position sensor was developed for measuring vibratory accelerations. The interferometer consists of interdigitated fingers that are alternately attached to the proof mass and support substrate. Illuminating the fingers with coherent light generates a series of diffracted beams. The intensity of a given beam depends on the out-of-plane separation between the proof mass fingers and support fingers. Proof masses with mechanical resonances ranging from 80 Hz to 1 kHz were fabricated with a two mask process involving two deep reactive ion etches, an oxide etch stop, and a polyimide protective layer. The structures were packaged with a laser diode and photodiode into 8.6-[cm.sup.3] acrylic housings. The 80-Hz resonant proof mass has a noise equivalent acceleration of 40 ng/rt Hz and a dynamic range of 85 dB at 40 Hz. [730]

The effect of mode number on energy dissipation in the microfluidic beam resonators is examined. The studies have shown that the quality factor has decreased with increasing mode number.