Michael G. Roper

A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in–answer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 μl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

Roles of Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, and Release of Intracellular Ca2+ Stores in Insulin-stimulated Insulin Secretion in β-Cells

The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca2+ concentration ([Ca2+] i ) in isolated mouse pancreatic β-cells and clonal β-cells was investigated. Application of insulin to single β-cells resulted in increases in [Ca2+] i that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca2+] i originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca2+] i changes and insulin secretion at single β-cells were abolished by treatment with 100 nm wortmannin or 1 μm thapsigargin; however, they were unaffected by 10 μm U73122, 20 μmnifedipine, or removal of Ca2+ from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 μm bisindolylmaleimide I, but [Ca2+] i changes were unaffected. In an insulin receptor substrate-1 gene disrupted β-cell tumor line, insulin did not evoke either [Ca2+] i changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca2+] i are due to release of intracellular Ca2+ stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca2+] i and activation of protein kinase C.

CCAAT/Enhancer-binding Protein β Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Leprdb/db Mice

CCAAT/enhancer-binding protein β (C/EBPβ) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPβ in hepatic lipogenesis remains undefined. Here we show that C/EBPβ inactivation in Leprdb/db mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPβ-/- x Leprdb/db mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPβ deletion in Leprdb/db mice down-regulated peroxisome proliferator-activated receptor γ2 (PPARγ2) and stearoyl-CoA desaturase-1 and up-regulated PPARα independent of SREBP1c. Conversely, C/EBPβ overexpression in wild-type mice increased PPARγ2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPβ or C/EBPβ RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARγ2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPβ expression in hepatocytes, whereas fatty acids up-regulate C/EBPβ expression. These data provide novel evidence linking C/EBPβ expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease.

Recent advances in microfluidic detection systems

There are numerous detection methods available for microfluidic analyses. Both conventional and novel detection methods are being put to use for detection on these miniaturized systems, with the analyte of interest driving the choice of detection method. In this article, we summarize microfluidic-based detection strategies from the last 2 years. More focus is given to unconventional approaches to detection routes and novel strategies for performing high-sensitivity detection.

Microfluidics-to-Mass Spectrometry: A review of coupling methods and applications

Microfluidic devices offer great advantages in integrating sample processes, minimizing sample and reagent volumes, and increasing analysis speed, while mass spectrometry detection provides high information content, is sensitive, and can be used in quantitative analyses. The coupling of microfluidic devices to mass spectrometers is becoming more common with the strengths of both systems being combined to analyze precious and complex samples. This review summarizes select achievements published between 2010 and July 2014 in novel coupling between microfluidic devices and mass spectrometers. The review is subdivided by the types of ionization sources employed, and the different microfluidic systems used.

Online coupling of digital microfluidic devices with mass spectrometry detection using an eductor with electrospray ionization.

MS detection coupled with digital microfluidic (DMF) devices has most commonly been demonstrated in an offline manner using matrix assisted laser desorption ionization. In this work, an eductor is demonstrated which facilitated online coupling of DMF with electrospray ionization MS detection. The eductor consisted of a transfer capillary, a standard ESI needle, and a tapered gas nozzle. As a pulse of N(2) was applied to the nozzle, a pressure differential was induced at the outlet of the ESI needle that pulled droplets from the DMF, past the ESI needle, and into the flow of gas exiting the nozzle, allowing detection by MS. Operating position, ionization potential, and N(2) pressure were optimized, with the optimum ionization potential and N(2) pressure found to be 3206 V and 80 psi, respectively. Online MS detection was demonstrated from both open and closed DMF devices using 2.5 μL and 630 nL aqueous droplets, respectively. Relative quantitation by DMF-MS was demonstrated by mixing droplets of caffeine with droplets of theophylline on an open DMF device and comparing the peak area ratio obtained to an on-chip generated calibration curve. This eductor-based method for transferring droplets has the potential for rapid, versatile, and high-throughput microfluidic analyses.

Microfluidic perfusion system for automated delivery of temporal gradients to islets of Langerhans

A microfluidic perfusion system was developed for automated delivery of stimulant waveforms to cells within the device. The 3-layer glass/polymer device contained two pneumatic pumps, a 12 cm mixing channel, and a 0.2 microL cell chamber. By altering the flow rate ratio of the pumps, a series of output concentrations could be produced while a constant 1.43 +/- 0.07 microL/min flow rate was maintained. The output concentrations could be changed in time producing step gradients and other waveforms, such as sine and triangle waves, at different amplitudes and frequencies. Waveforms were analyzed by comparing the amplitude of output waveforms to the amplitude of theoretical waveforms. Below a frequency of 0.0098 Hz, the output waveforms had less than 20% difference than input waveforms. To reduce backflow of solutions into the pumps, the operational sequence of the valving program was modified, as well as differential etching of the valve seat depths. These modifications reduced backflow to the point that it was not detected. Gradients in glucose levels were applied in this work to stimulate single islets of Langerhans. Glucose gradients between 3 and 20 mM brought clear and intense oscillations of intracellular [Ca(2+)] indicating the system will be useful in future studies of cellular physiology.

Microfluidic System for Generation of Sinusoidal Glucose Waveforms for Entrainment of Islets of Langerhans

A microfluidic system was developed to produce sinusoidal waveforms of glucose to entrain oscillations of intracellular [Ca(2+)] in islets of Langerhans. The work described is an improvement to a previously reported device where two pneumatic pumps delivered pulses of glucose and buffer to a mixing channel. The mixing channel acted as a low pass filter to attenuate these pulses to produce the desired final concentration. Improvements to the current device included increasing the average pumping frequency from 0.83 to 3.33 Hz by modifying the on-chip valves to minimize adhesion between the PDMS and glass within the valve. The cutoff frequency of the device was increased from 0.026 to 0.061 Hz for sinusoidal fluorescein waves by shortening the length of the mixing channel to 3.3 cm. The value of the cutoff frequency was chosen between the average pumping frequency and the low frequency (approximately 0.0056 Hz) glucose waves that were needed to entrain the islets of Langerhans. In this way, the pulses from the pumps were attenuated greatly but the low-frequency glucose waves were not. With the use of this microfluidic system, a total flow rate of 1.5 +/- 0.1 microL min(-1) was generated and used to deliver sinusoidal waves of glucose concentration with a median value of 11 mM and amplitude of 1 mM to a chamber that contained an islet of Langerhans loaded with the Ca(2+)-sensitive fluorophore, indo-1. Entrainment of the islets was demonstrated by pacing the rhythm of intracellular [Ca(2+)] oscillations to oscillatory glucose levels produced by the device. The system should be applicable to a wide range of cell types to aid understanding cellular responses to dynamically changing stimuli.

Transport and separation of biomolecular cargo on paramagnetic colloidal particles in a magnetic ratchet.

Paramagnetic particles in a magnetic ratchet potential were transported in discrete steps in an aqueous solution on the surface of a magnetic garnet film. The proposed technique allows the simultaneously controlled, dispersion-free movement of an ensemble of paramagnetic particles across the surface. External magnetic modulations were used to transport the particles in a defined direction, and a current reversal upon changing the size of the particles was used to separate particles having different diameters. Doublets consisting of a larger and a smaller particle functionalized with complimentary oligonucleotides and bound via Watson-Crick base pairing were separated after melting the double stranded DNA.

Glass microfluidic devices with thin membrane voltage junctions for electrospray mass spectrometry

In this study a novel glass membrane was prepared for conducting high voltage (HV) to solution in the channel of a microfabricated device for generation of liquid electrospray. Taylor cone formation and mass spectra obtained from this microdevice confirmed the utility of the glass membrane, but voltage conduction through the membrane could not be successfully explained based solely on the conductivity of the glass itself. This novel method for developing a high-voltage interface for microdevices avoids direct metal/liquid contact eliminating bubble formation in the channel due to water hydrolysis on the surface of the metal. Further, this arrangement produces no dead volume as is often found with traditional liquid junctions. At the same time, preliminary investigations into the outlet design of glass microdevices for interfacing with electrospray mass spectrometry, was explored. Both the exit shape and the use of hydrophobic coatings at the channel exit of the microdevice electrospray interface were evaluated using standard proteins with results indicating the utility of this type of design after further optimization.