Microscale isoelectric fractionation using photopolymerized membranes.

In this work, we introduce microscale isoelectric fractionation (µIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. µIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.

On-chip isoelectric focusing using photopolymerized immobilized pH gradients.

We present the first successful adaptation of immobilized pH gradients (IPGs) to the microscale (muIPGs) using a new method for generating precisely defined polymer gradients on-chip. Gradients of monomer were established via diffusion along 6 mm flow-restricted channel segments. Precise control over boundary conditions and the resulting gradient is achieved by continuous flow of stock solutions through side channels flanking the gradient segment. Once the desired gradient is established, it is immobilized via photopolymerization. Precise gradient formation was verified with spatial and temporal detection of a fluorescent dye added to one of the flanking streams. Rapid (<20 min) isoelectric focusing of several fluorescent pI markers and proteins is demonstrated across pH 3.8-7.0 muIPGs using both denaturing and nondenaturing conditions, without the addition of carrier ampholytes. The muIPG format yields improved stability and comparable resolution to prominent on-chip IEF techniques. In addition to rapid, high-resolution separations, the reported muIPG format is amenable to multiplexed and multidimensional analysis via custom gradients as well as integration with other on-chip separation methods.

Integrated microfluidic platform for oral diagnostics.

While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3-10 min) with low sample volume requirements (10 microL) and appreciable sensitivity (nM-pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip-based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

An embedded microretroreflector-based microfluidic immunoassay platform.

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 µm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.

Controlled microfluidic reconstitution of functional protein from an anhydrous storage depot.

A novel method has been developed for preserving molecules in microfluidic devices that also enables the control of the spatial and temporal concentrations of the reconstituted molecules within the devices. In this method, a storage cavity, embedded in a microchannel, is filled with a carbohydrate matrix containing, for example, a reagent. When the matrix is exposed to flowing liquid, it dissolves, resulting in the controlled reconstitution and release of the reagent from the cavity. The technique was demonstrated using two different model systems; the successful preservation and controlled release of beta-galactosidase was achieved. This method has possible applications for simple point-of-care drug delivery and immunoassays, and could be used to pattern the surfaces of microchannels. More broadly, this preservation and controlled release technique can be applied where the preservation and/or spatial and temporal control of chemical concentrations are desired.

Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels.

We present advancements in microfluidic technology for rapid detection of as few as 10 rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (min), high-efficiency (~100%) capture. In the process, targeted cells in sample volumes of 10 µl to >100 µl are concentrated within a sub-50 nl region at the PAM filter edge in the microchannel, thus concentrating them over 1000-fold. This significantly increases sensitivity, as the hydrophilic PAM also yields low non-specific immuno-fluorescence backgrounds with samples including serum, blood and non-targeted bacteria. The concentrated target cells are detected using fluorescently-labeled antibodies. With a single 2.0×2.0×0.3 mm PAM filter, as few as 10 rickettsial organisms per 100 µl of lysed blood sample can be analyzed within 60 min, as compared to hours or even days needed for conventional detection methods. This method is highly relevant to rapid, multiplexed, low-cost point of care diagnostics at early stages of infection where diagnostics providing more immediate and actionable test results are needed to improve patient outcomes and mitigate potential natural and non-natural outbreaks or epidemics of rickettsial diseases.

Thirty-minute total synthesis of microfluidic systems and functionalized porous elements via "living" radical photo-polymerization.

A "living" radical photo-polymerization (LRPP) technique is used to rapidly fabricate microfluidic channels and micro-patterned porous polymer monoliths. Surface-initiated LRPP is then used to functionalize porous elements in a robust one-step surface modification process. Assay-ready platforms can be fully realized in less than 30 minutes. An application relevant to clinical diagnostics is presented.

Microfluidic immunoassays as rapid saliva-based clinical diagnostics.

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 microl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.

Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels.

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics.