Isoelectric trapping and discrimination of histones from plasma in a microfluidic device using dehydrated isoelectric gate.

Histones are basic proteins with an isoelectric point around 11. It has been shown that the level of plasma circulating histones increases significantly during sepsis, and circulating free histones are associated with sepsis severity and mortality. It was found that the median plasma total free histone concentration of sepsis ICU non-survivors is higher compared to survivors. Therefore, histone concentration can serve as a prognostic indicator and there is a need for a simple, low-cost, and rapid method for measuring histone levels. In this work, we have developed a microfluidic device containing an isoelectric membrane made of dehydrated agarose gel of a specific pH embedded in a porous membrane for isoelectric trapping of histones rapidly. Although isoelectric gates have been used for trapping proteins before, they have to be introduced at the time of the experiment. Here, we show that isoelectric gates formed by gels loaded in a scaffold can be integrated directly into the fabrication process flow, dehydrated for storage, and rehydrated during the experiment and still function effectively to achieve isoelectric trapping. A low-cost and rapid microfabrication technique, xurography, was used for agarose integration and device fabrication. The integrated device was tested with samples containing buffered histone, histone in the presence of high-concentration bovine serum albumin (BSA), and histone spiked in blood plasma. The results show that the device can be used to distinguish between survivors and non-survivors of sepsis in less than 10 min, making it suitable as a point-of-care device for sepsis prognosis.

Microfluidic device for single step measurement of protein C in plasma samples for sepsis prognosis.

Protein C is a vitamin K dependant protein in plasma that plays an essential role in regulating the coagulation cascade and inflammatory response. As a result of its importance in these roles, it has been suggested as a biomarker for prognosis of patients affected by sepsis. Sepsis is a dysregulated host response to an infection that is the leading cause of mortality in U.S. hospitals and results in the highest cost of hospitalization. It was found that protein C concentration in non-surviving sepsis patients is significantly lower (1.8 µg mL-1) than in survivors and healthy patients who have a protein C concentration of 3.9-5.9 µg mL-1. Current methods for diagnosing sepsis rely on expensive immunoassays or functional assays that require multiple steps for isolation and activation of protein C. We demonstrate in this paper a low cost, single step assay for detection of protein C in blood plasma. This was done by combining isoelectric gates with barium-immobilized metal affinity trapping. The electric field was optimized for use with immobilized metal affinity using COMSOL simulation. The integrated device was tested with samples containing buffered protein C, protein C in the presence of high concentration bovine serum albumin and alpha 1-proteinase inhibitor, and in blood plasma with spiked protein C. The stability of the measured values was tested by monitoring the intensity of a mixture of protein C with BSA and A1PI every minute to determine that measurement after 40 minutes was optimal. The results showed that the device could be used to distinguish a reduction in protein C from 4.46 µg mL-1 to 1.96 µg mL-1 with greater than 98% confidence in plasma making it suitable for sepsis prognosis.

Single-step measurement of cell-free DNA for sepsis prognosis using a thread-based microfluidic device.

Cell-free DNA (cfDNA) content in plasma has been studied as a biomarker for sepsis. Recent publications show that the cfDNA content in sepsis patients entering intensive care unit who were likely to survive had a total cfDNA concentration of 1.16 ± 0.13 µg/mL compared to 4.65 ± 0.48 µg/mL of non-survivors. Current methods for measuring cfDNA content in plasma were designed to amplify and measure low concentrations of specific DNA, making them unsuitable for low-cost measurement of total cfDNA content in plasma. Here, we have developed a point of care (POC) device that uses a thread silicone device as a medium to store a fluorescent dye which eliminates the need for preparatory steps, external aliquoting and dispensing of reagents, preconcentration, and external mixing while reducing the detection cost. The device was paired with a portable imaging system with an excitation filter at 472 ± 10 nm and an emission filter of 520 ± 10 nm that can be operated with just 100 mA current supply. The device was demonstrated for use in the quantification of buffered cfDNA samples in a range 1-6 µg/mL with a sensitivity of 5.72 AU/µg/mL and with cfDNA spiked in plasma with a range of 1-3 µg/mL and a sensitivity of 5.43 AU/µg/mL. The results showed that the device could be used as a low-cost, rapid, and portable POC device for differentiating between survivors and non-survivors of sepsis within 20 min.

Potential role of blood biomarkers in patients with fibromyalgia: a systematic review with meta-analysis.

ABSTRACT: Fibromyalgia (FM) is a complex chronic pain condition. Its symptoms are nonspecific, and to date, no objective test exists to confirm FM diagnosis. Potential objective measures include the circulating levels of blood biomarkers. This systematic review and meta-analysis aim to review studies assessing blood biomarkers' levels in patients with FM compared with healthy controls. We systematically searched the PubMed, MEDLINE, EMBASE, and PsycINFO databases. Fifty-four studies reporting the levels of biomarkers in blood in patients with FM were included. Data were extracted, and the methodological quality was assessed independently by 2 authors. The methodological quality of 9 studies (17%) was low. The results of most studies were not directly comparable given differences in methods and investigated target immune mediators. Thus, data from 40 studies only were meta-analyzed using a random-effects model. The meta-analysis showed that patients with FM had significantly lower levels of interleukin-1 ß and higher levels of IL-6, IL-8, tumor necrosis factor-alpha, interferon gamma, C-reactive protein, and brain-derived neurotrophic factor compared with healthy controls. Nevertheless, this systematic literature review and meta-analysis could not support the notion that these blood biomarkers are specific biomarkers of FM. Our literature review, however, revealed that these same individual biomarkers may have the potential role of identifying underlying pathologies or other conditions that often coexist with FM. Future research is needed to evaluate the potential clinical value for these biomarkers while controlling for the various confounding variables.