Determination of partition coefficients of biomolecules in a microfluidic aqueous two phase system platform using fluorescence microscopy.

Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in molecular solubility in each of the two immiscible phases. However, ATPS use has been limited due to the difficulty in predicting the behavior of a given biomolecule in the partition environment together with the empirical and time-consuming techniques that are used for the determination of partition and extraction parameters. In this work, a fast and novel technique based on a microfluidic platform and using fluorescence microscopy was developed to determine the partition coefficients of biomolecules in different ATPS. This method consists of using a microfluidic device with a single microchannel and three inlets. In two of the inlets, solutions containing the ATPS forming components were loaded while the third inlet was fed with the FITC tagged biomolecule of interest prepared in milli-Q water. Using fluorescence microscopy, it was possible to follow the location of the FITC-tagged biomolecule and, by simply varying the pumping rates of the solutions, to quickly test a wide variety of ATPS compositions. The ATPS system is allowed 4min for stabilization and fluorescence micrographs are used to determine the partition coefficient.The partition coefficients obtained were shown to be consistent with results from macroscale ATPS partition. This process allows for faster screening of partition coefficients using only a few microliters of material for each ATPS composition and is amenable to automation. The partitioning behavior of several biomolecules with molecular weights (MW) ranging from 5.8 to 150kDa, and isoelectric points (pI) ranging from 4.7 to 6.4 was investigated, as well as the effect of the molecular weight of the polymer ATPS component.

The application of microbeads to microfluidic systems for enhanced detection and purification of biomolecules.

This paper describes microbead-based microfluidic systems. Several aspects of bead assays in microfluidics make them advantageous for bioassays in simple microchannels, including enhanced surface-to-volume ratio, improved molecular recognition reaction efficiency, and the wide range of surface functionalization available with commercial microbeads. Two-level SU-8 molds are used to fabricate PDMS microchannels that can hydrodynamically trap different types of microbeads, with characteristic dimensions of tens of microns. The use of these microbead-based microfluidic systems in the biosensing of antibodies, toxins and nucleic acids, as well as in antibody purification will be presented and discussed in this paper.

A point-of-use microfluidic device with integrated photodetector array for immunoassay multiplexing: Detection of a panel of mycotoxins in multiple samples.

For a point-of-use analytical device to be successful in real-world applications, it needs to be rapid, simple to operate and, ideally, able to multiplex the detection of several analytes and samples. Mycotoxin detection in food and feedstock in particular has become increasingly relevant as these toxins, such as ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON), are subject to strict regulations and recommendations in the European Union. A novel, simple, negative pressure-driven device with manually operated magnetic valves was developed and the simultaneous immunodetection of these three mycotoxins was demonstrated via the laminar flow patterning of probes in an area of ≈0.12mm2 and subsequent chemiluminescence generation via HRP-labeled antibodies. The three mycotoxins were detected in less than 20min at concentrations of 100ng/mL for OTA and DON and 3ng/mL for AFB1, spiked in a sample under analysis and simultaneously compared to a toxin-free reference and a standard contaminated with critical target concentrations. The on-chip optical detection was performed in a single acquisition step by integrating a microfabricated array of 25×25µm2 hydrogenated amorphous silicon (a-Si:H) photosensors below the microfluidic chip. The device presented in this work is simple and effective for point-of-use multiplexing of immunoassays and was applied in this work to the screening of mycotoxins.

Study on the bio-functionalization of memristive nanowires for optimum memristive biosensors.

Semiconductor nanowires are emerging as promising building blocks for biosensors enabling direct electrical detection of various biomolecules. In this framework, two-terminal Schottky-barrier silicon (Si) nanowire arrays that exhibit memristive electrical response, so-called memristive devices, are bio-functionalized and converted to memristive biosensors for bio-detection purposes. A comparative analysis of three bio-functionalization strategies is proposed here in order to design and develop optimum memristive biosensors to be implemented in label-free sensing applications. The surface of the device is modified with an anti-free-Prostate Specific Antigen (PSA) antibody as the case of study via: (a) direct adsorption on the device surface, (b) a bio-affinity approach using biotin-streptavidin combination and (c) covalent attachment using (3-glycidyloxypropyl)trimethoxysilane (GPTES). The optimum memristive biosensor is defined via the calibration and comparative study of the biosensors' electrical response under controlled environmental conditions (humidity and temperature) in order to maximize the performance of the biosensor. In addition, it is demonstrated that the direct passive adsorption strategy presents double the performance of the other two methods. The uptake of biological molecules on the nanostructure surface is verified by atomic force microscopy and confocal microscopy. Scanning electron microscopy reveals the details of the surface morphology of the nanofabricated structures before and after bio-functionalization for the three methods applied. The system shows potential for general application in molecular diagnostics, and, in particular, for the early detection of prostate cancer.

Determination of aqueous two phase system binodal curves using a microfluidic device.

Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in solubility in each of the two phases. However, their use has been greatly hindered due to poor theoretical understanding of the principles behind ATPS formation and the empirical and time-consuming techniques used for the determination of optimal extraction parameters including the binodal curves. In this work, characteristic ATPS binodal curves were determined by a novel technique in which the formation of an ATPS system is measured in a microfluidic device. Two solutions containing separate ATPS solution precursors were loaded into the side inlets of a three inlet microfluidic channel while milli-Q water was loaded into the middle inlet. By varying the flow rates of the three solutions, a wide range of concentrations inside the microchannel could be rapidly tested using limited volumes. Using optical microscopy, depending on the concentrations inside the microchannel, three different states could be observed at the end of the microchannel (i) the presence of an interface; (ii) no presence of an interface; or (iii) the presence of an unstable interface. The binodal curve was calculated using the points corresponding to unstable interfaces and compared to binodal curves obtained through the standard turbidometric titration method for both PEG/salt and PEG/dextran systems.

On-chip sample preparation and analyte quantification using a microfluidic aqueous two-phase extraction coupled with an immunoassay.

Immunoassays are fast and sensitive techniques for analyte quantification, and their use in point-of-care devices for medical, environmental, and food safety applications has potential benefits of cost, portability, and multiplexing. However, immunoassays are often affected by matrix interference effects, requiring the use of complex laboratory extraction and concentration procedures in order to achieve the required sensitivity. In this paper we propose an integrated microfluidic device for the simultaneous matrix clean-up, concentration and detection. This device consists of two modules in series, the first performing an aqueous two-phase extraction (ATPE) for matrix extraction and analyte pre-concentration, and the second an immunoassay for quantification. The model analyte was the mycotoxin ochratoxin A (OTA) in a wine matrix. Using this strategy, a limit of detection (LoD) of 0.26 ng mL(-1) was obtained for red wine spiked with OTA, well below the regulatory limit for OTA in wines of 2 ng mL(-1) set by the European Union. Furthermore, the linear response on the logarithmic concentration scale was observed to span 3 orders of magnitude (0.1-100 ng mL(-1)). These results are comparable to those obtained for the quantification of OTA in plain buffer without an integrated ATPE (LoD = 0.15 ng mL(-1)). The proposed method was also found to provide similar results for markedly different matrices, such as red and white wines. This novel approach based on aqueous two-phase systems can help the development of point-of-care devices that can directly deal with real samples in complex matrices without the need for extra extraction processes and equipment.

Aqueous two-phase systems for enhancing immunoassay sensitivity: simultaneous concentration of mycotoxins and neutralization of matrix interference.

Immunoassays have a broad application range, from environmental and food toxicology to biomedical analysis, providing rapid and simple methods for analyte quantification. Immunoassays, however, are often challenging at nM and sub nM concentrations and are affected by detrimental matrix interference effects, as is the case of the detection of ochratoxin A (OTA) and Aflatoxin B1 (AFB1). These are widespread mycotoxins found in food and feed, with serious potential implications for human health. This work demonstrates the use of polymer-salt aqueous two phase systems (ATPSs) for the simultaneous concentration of mycotoxins and neutralization of matrix interference. In particular, polyethylene glycol (PEG)-phosphate salt ATPSs were used to enhance the detection sensitivity of OTA and AFB1 in wines and beer by an indirect competitive ELISA. Using this methodology it was possible to quantify both analytes spiked in red wine with limits-of-detection (LoD) down to 0.19 ng/mL and 0.035 ng/mL, respectively, with results comparable to those obtained using solutions of toxins in phosphate buffered saline (PBS) buffer (0.7 ng/mL and 0.009 ng/mL, respectively). Furthermore, a very low matrix-to matrix variability was observed, with LoD and half inhibitory concentration (IC50) values of 5.17 ± 1.08 and 33.2 ± 3.5 ng/mL (±SD) obtained in the detection of OTA spiked in red and white wines, beer or PBS buffer. These results indicate the potential of ATPS as a fast and simple concentration step and in providing matrix-independent analyte quantification for enhanced immunoassay sensitivity below regulatory levels.

Integrated optical detection of autonomous capillary microfluidic immunoassays:a hand-held point-of-care prototype.

The miniaturization of biosensors using microfluidics has potential in enabling the development of point-of-care devices, with the added advantages of reduced time and cost of analysis with limits-of-detection comparable to those obtained through traditional laboratory techniques. Interfacing microfluidic devices with the external world can be difficult especially in aspects involving fluid handling and the need for simple sample insertion that avoids special equipment or trained personnel. In this work we present a point-of-care prototype system by integrating capillary microfluidics with a microfabricated photodiode array and electronic instrumentation into a hand-held unit. The capillary microfluidic device is capable of autonomous and sequential fluid flow, including control of the average fluid velocity at any given point of the analysis. To demonstrate the functionality of the prototype, a model chemiluminescence ELISA was performed. The performance of the integrated optical detection in the point-of-care prototype is equal to that obtained with traditional bench-top instrumentation. The photodiode signals were acquired, displayed and processed by a simple graphical user interface using a computer connected to the microcontroller through USB. The prototype performed integrated chemiluminescence ELISA detection in about 15 min with a limit-of-detection of ≈2 nM with an antibody-antigen affinity constant of ≈2×10(7) M(-1).

Monitoring intracellular calcium in response to GPCR activation using thin-film silicon photodiodes with integrated fluorescence filters.

G-protein coupled receptor (GPCRs) drug discovery is a thriving strategy in the pharmaceutical industry. The standard approach uses living cells to test millions of compounds in a high-throughput format. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluorescence or luminescence read-out systems, which require external equipment for signal acquisition. In this work, thin-film amorphous silicon photodiodes with an integrated fluorescence filter were developed to capture the intracellular calcium dynamics in response to the activation of the endogenous muscarinic M1 GPCR of HEK 293T cells. Using the new device it was possible to characterize the potency of carbachol (EC50=10.5 µM) and pirenzepine (IC50=4.2 µM), with the same accuracy as standard microscopy optical systems. The smaller foot-print provided by the detection system makes it an ideal candidate for the future integration in microfluidic devices for drug discovery.

The effect of the surface functionalization and the electrolyte concentration on the electrical conductance of silica nanochannels.

It is known that the conductance of nanochannels as a function of electrolyte concentration deviates from a linearly proportional relationship and approaches a value independent of the concentration as the electrolyte concentration is lowered. Most of the proposed models account for this behavior by considering a constant surface charge density and an ideal electrolyte solution. However, at low electrolyte concentrations, the ideal electrolyte approximation is no longer valid because the ions that result from the atmospheric carbon dioxide dissolution in water dominate the ionic concentration. In this paper, arrays of silica nanochannels were electrically characterized via conductance measurements. The conductance at low salt concentrations is modeled by a variable surface charge model that accounts for all ionic species in solution. This model was used to determine the variable surface charge of the bare silica nanochannels as well as of chemically modified nanochannels. The model correctly predicted the variation of the nanochannel conductance observed after silane (aminopropyldimethylethoxysilane) functionalization and single-strand DNA immobilization. Finally, pH modification of bulk KCl solutions was employed as an alternative method of changing the surface charge of silica nanochannels. Surface charge calculated from conductance measurements performed at different bulk pH values confirmed that the surface charge of the silica nanochannel walls is sensitive to the H(+) concentration.

Design of a microfluidic platform for monoclonal antibody extraction using an aqueous two-phase system.

The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14-16.8 cm) and flow rates of the salt (1-2 µL/min) and PEG-rich phases (0.2-0.5 µL/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization.

Integrated detection of intrinsic fluorophores in live microbial cells using an array of thin film amorphous silicon photodetectors.

Two-dimensional fluorescence spectroscopy (2D FS) provides a non-invasive means to assess cell condition without the introduction of changes to the cell environment. The method relies on the measurement of the excitation-emission fluorescence intensity matrix of key intrinsic fluorophores, like aromatic amino acids, enzyme cofactors, and vitamins. Commonly used detection systems are complex, with multiple bandpass filters, and are hard to miniaturize. Here, an amorphous silicon photodetector array system integrated with amorphous silicon-carbon alloy filters designed to detect three key fluorophores - tryptophan (Trp), reduced nicotine adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) - is demonstrated. These intrinsic fluorophores were detected in pure solutions and also in suspended yeast cells. The array system was used to monitor changes in intrinsic fluorophore concentration when a yeast cell solution was subject to a thermal shock stress.

Heterogeneous immunoassays in microfluidic format using fluorescence detection with integrated amorphous silicon photodiodes.

Miniaturization of immunoassays through microfluidic technology has the potential to decrease the time and the quantity of reactants required for analysis, together with the potential of achieving multiplexing and portability. A lab-on-chip system incorporating a thin-film amorphous silicon (a-Si:H) photodiode microfabricated on a glass substrate with a thin-film amorphous silicon-carbon alloy directly deposited above the photodiode and acting as a fluorescence filter is integrated with a polydimethylsiloxane-based microfluidic network for the direct detection of antibody-antigen molecular recognition reactions using fluorescence. The model immunoassay used consists of primary antibody adsorption to the microchannel walls followed by its recognition by a secondary antibody labeled with a fluorescent quantum-dot tag. The conditions for the flow-through analysis in the microfluidic format were defined and the total assay time was 30 min. Specific molecular recognition was quantitatively detected. The measurements made with the a-Si:H photodiode are consistent with that obtained with a fluorescence microscope and both show a linear dependence on the antibody concentration in the nanomolar-micromolar range.

The effect of the shape of single, sub-ms voltage pulses on the rates of surface immobilization and hybridization of DNA.

Electric fields generated by single square and sinusoidal voltage pulses with amplitudes below 2 V were used to assist the covalent immobilization of single-stranded, thiolated DNA probes, onto a chemically functionalized SiO2 surface and to assist the specific hybridization of single-stranded DNA targets with immobilized complementary probes. The single-stranded immobilized DNA probes were either covalently immobilized (chemisorption) or electrostatically adsorbed (physisorption) to a chemically functionalized surface. Comparing the speed of electric field assisted immobilization and hybridization with the corresponding control reactions (without electric field), an increase of several orders of magnitude is observed, with the reaction timescaled down from 1 to 2 h to a range between 100 ns and 1 ms. The influence of the shape of the voltage pulse (square versus sinusoidal) and its duration were studied for both immobilization and hybridization reactions. The results show that pulsed electric fields are a useful tool to achieve temporal and spatial control of surface immobilization and hybridization reactions of DNA.

Detection of DNA and proteins using amorphous silicon ion-sensitive thin-film field effect transistors.

Amorphous silicon-based ion-sensitive field-effect transistors (a-Si:H ISFETs) are used for the label-free detection of biological molecules. The covalent immobilization of DNA, followed by DNA hybridization, and of the surface adsorption of oligonucleotides and proteins were detected electronically by the a-Si:H ISFET. The ISFET measurements are performed with an external Ag/AgCl microreference electrode immersed in 100mM phosphate buffer electrolyte with pH 7.0. Threshold voltage shifts in the transfer curve of the ISFETs are observed resulting from successive steps of surface chemical functionalization, covalent DNA attachment to the functionalized surface, surface blocking, and hybridization with a complementary target. The surface sensitivity achieved for DNA oligonucleotides is of the order of 1pmol/cm(2). Point-of-zero charge estimations were made for the functionalized surfaces and for the device surface after DNA immobilization and hybridization. The results show a correlation between the changes in the point-of-zero charge and the shift observed in the threshold voltage of the devices. Electronic detection of adsorbed proteins and DNA is also achieved by monitoring the shifts of the threshold voltage of the ISFETs, with a sensitivity of approximately 50nM.

Single base mismatch detection by microsecond voltage pulses.

A single square voltage pulse applied to metal electrodes underneath a silicon dioxide film upon which DNA probes are immobilized allows the discrimination of DNA targets with a single base mismatch during hybridization. Pulse duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of electric field assisted hybridization. Although pulses with 1 V, lasting less than 1 ms and with a rise/fall times of 4.5 ns led to maximum hybridization of fully complementary strands, lack of stringency did not allow the discrimination of single base mismatches. However, by choosing pulse conditions that are slightly off the optimum, the selectivity for discriminating single base mismatches could be improved up to a factor approximately 5 when the mismatch was in the middle of the strand and up to approximately 1.5 when the mismatch was on the 5'-end and. These results demonstrate that hybridization with the appropriate electric field pulse provides a new, site-specific, approach to the discrimination of single nucleotide polymorphisms in the sub-millisecond time scale, for addressable DNA microarrays.

Electric-field assisted immobilization and hybridization of DNA oligomers on thin-film microchips.

Single, square voltage pulses in the microsecond timescale result in selective 5'-end covalent bonding (immobilization) of thiolated single-stranded (ss) DNA probes to a modified silicon dioxide flat surface and in specific hybridization of ssDNA targets to the immobilized probe. Immobilization and hybridization rates using microsecond voltage pulses at or below 1 V are at least 10(8) times faster than in the passive control reactions performed without electric field (E), and can be achieved with at least three differently functionalized thin-film surfaces on plastic or glass substrates. The systematic study of the effect of DNA probe and target concentrations, of DNA probe and target length, and the application of asymmetric pulses on E-assisted DNA immobilization and hybridization showed that: (1) the rapidly rising edge of the pulse is most critical to the E-assisted processes, but the duration of the pulse is also important; (2) E-assisted immobilization and hybridization can be performed with micrometre-sized pixels, proving the potential for use on microelectronic length scales, and the applied voltage can be scaled down together with the electrode spacing to as low as 25 mV; and (3) longer DNA chains reduce the yield in the E-assisted immobilization and hybridization because the density of physisorbed single-stranded DNA is reduced. The results show that the E-induced reactions can be used as a general method in DNA microarrays to produce high-density DNA chips (E-immobilization) and speed the microarray-based analysis (E-hybridization).

An on-chip thin film photodetector for the quantification of DNA probes and targets in microarrays.

A flat microdevice which incorporates a thin-film amorphous silicon (a-Si:H) photodetector with an upper layer of functionalized SiO2 is used to quantify the density of both immobilized and hybridized DNA oligonucleotides labeled with a fluorophore. The device is based on the photoconductivity of hydrogenated amorphous silicon in a coplanar electrode configuration. Excitation, with near UV/blue light, of a single-stranded DNA molecule tagged with the fluorophore 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide (PyMPO), results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the optoelectronic detection of the DNA molecules. The detection limit of the present device is of the order of 1 x 10(12) molecules/cm2 and is limited by the efficiency of the filtering of the excitation light. A surface density of 33.5 +/- 4.0 pmol/cm2 was measured for DNA covalently immobilized to the functionalized SiO2 thin film and a surface density of 3.7 +/- 1.5 pmol/cm2 was measured for the complementary DNA hybridized to the bound DNA. The detection concept explored can enable on-chip electronic data acquisition, improving both the speed and the reliability of DNA microarrays.

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays.

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO(2) surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 micros, respectively, about 10(9) times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.