Rapid diagnosis of bloodstream infections using a culture-free phenotypic platform.

BACKGROUND: Bloodstream infections (BSIs) are a life-threatening acute medical condition and current diagnostics for BSIs suffer from long turnaround time (TAT). Here we show the validation of a rapid detection-analysis platform (RDAP) for the diagnosis of BSIs performed on clinical blood samples METHODS: The validation was performed on a cohort of 59 clinical blood samples, including positive culture samples, which indicated confirmed bloodstream infections, and negative culture samples. The bacteria in the positive culture samples included Gram-positive and Gram-negative pathogenic species. RDAP is based on an electrochemical sandwich immunoassay with voltage-controlled signal amplification, which provides an ultra-low limit of detection (4 CFU/mL), allowing the platform to detect and identify bacteria without requiring culture and perform phenotypic antibiotic susceptibility testing (AST) with only 1-2 h of antibiotic exposure. The preliminary diagnostic performance of RDAP was compared with that of standard commercial diagnostic technologies. RESULTS: Using a typical clinical microbiology laboratory diagnostic workflow that involved sample culture, agar plating, bacteria identification using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry, and AST using MicroScan as a clinical diagnostic reference, RDAP showed diagnostic accuracy of 93.3% and 95.4% for detection-identification and AST, respectively. However, RDAP provided results at least 15 h faster. CONCLUSIONS: This study shows the preliminary feasibility of using RDAP to rapidly diagnose BSIs, including AST. Limitations and potential mitigation strategies for clinical translation of the present RDAP prototype are discussed. The results of this clinical feasibility study indicate an approach to provide near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.

Effective treatment of bloodstream infections (BSIs), a life-threatening acute medical condition, requires rapid diagnosis. Current diagnostic methods involve culturing the bacteria from the patient's blood, which requires typically 16­48 h to produce a diagnosis. Here, we demonstrate the feasibility of using a culture-free platform to perform rapid diagnosis of BSIs. We tested the diagnostic platform on a cohort of clinical blood samples. The bacteria contained in the samples covered a representative range of bacteria that cause BSIs. The culture-free platform produced diagnosis in about 15 hours faster than standard commercial diagnostic technologies and  the diagnostic results were in good agreement with that of standard technologies. The results of this study indicate an approach to providing near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.

Detection of a Traumatic Brain Injury Biomarker at the 10 fg/mL Level.

BACKGROUND: The detection of minute amounts of protein biomarkers in body fluids is believed to provide early diagnosis and prognosis of mild traumatic brain injury (mTBI). An ultrasensitive detection method was used to detect S100B, the most studied potential marker for the diagnosis of mTBI. METHODS: The detection method was a modified electrochemical immunoassay technique that provides voltage controlled intrinsic current signal amplification. The sandwich immune complex of S100B was formed on the working electrode of the screen-printed electrode. The gating voltage provides amplification of the current signal that flows through the complex. RESULTS: S100B was spiked in human serum. The limit of detection of S100B in human serum was 10 fg/mL. The calibration curves cover four orders of magnitudes from 10 fg/mL to 10 ng/mL. The specificity of the detection was demonstrated using TAU protein, which is another marker for mTBI. CONCLUSION: The results reported in this work using the field effect enzymatic detection (FEED)-based immunoassay indicate the feasibility of using this method for the detection of extremely low concentrations of markers of mTBI in human serum. This method can be developed as a platform for a range of markers of mTBI.

Culture-free bacterial detection and identification from blood with rapid, phenotypic, antibiotic susceptibility testing.

The current culture-based approach for the diagnosis of bloodstreams infection is incommensurate with timely treatment and curbing the prevalence of multi-drug resistant organisms (MDROs) due to its long time-to-result. Bloodstream infections typically involve extremely low (e.g., <10 colony-forming unit (CFU)/mL) bacterial concentrations that require a labor-intensive process and as much as 72 hours to yield a diagnosis. Here, we demonstrate a culture-free approach to achieve rapid diagnosis of bloodstream infections. An immuno-detection platform with intrinsic signal current amplification was developed for the ultrasensitive, rapid detection, identification (ID) and antibiotic susceptibility testing (AST) of infections. With its capability of monitoring short-term (1-2 hours) bacterial growth in blood, the platform is able to provide 84-minute simultaneous detection and ID in blood samples below the 10 CFU/mL level and 204-minute AST. The susceptible-intermediate-resistant AST capacity was demonstrated.

Ultrasensitive Detection of Shigella Species in Blood and Stool.

A modified immunosensing system with voltage-controlled signal amplification was used to detect Shigella in stool and blood matrixes at the single-digit CFU level. Inactivated Shigella was spiked in these matrixes and detected directly. The detection was completed in 78 min. Detection limits of 21 CFU/mL and 18 CFU/mL were achieved in stool and blood, respectively, corresponding to 2-7 CFUs immobilized on the detecting electrode. The outcome of the detection of extremely low bacterium concentration, i.e., below 100 CFU/mL, blood samples show a random nature. An analysis of the detection probabilities indicates the correlation between the sample volume and the success of detection and suggests that sample volume is critical for ultrasensitive detection of bacteria. The calculated detection limit is qualitatively in agreement with the empirically determined detection limit. The demonstrated ultrasensitive detection of Shigella on the single-digit CFU level suggests the feasibility of the direct detection of the bacterium in the samples without performing a culture.

Enhanced ethanol production via electrostatically accelerated fermentation of glucose using Saccharomyces cerevisiae.

The global demand for ethanol as an alternative fuel continues to rise. Advancement in all aspects of ethanol production is deemed beneficial to the ethanol industry. Traditional fermentation requires 50-70 hours to produce the maximum ethanol concentration of 7-8% (v/v). Here we demonstrate an electrostatic fermentation method that is capable of accelerating the fermentation of glucose using generic Saccharomyces cerevisiae as the fermenting microorganism to produce ethanol. The method, when applied to the batch fermentation of 1 liter fermenting mixture containing dry yeast without pre-culture, is able to achieve ethanol yield on the high gravity level (12.3% v/v) in 24 hours. The fermentation results in almost complete consumption of glucose. With pre-cultured yeast, ethanol yield can reach 14% v/v in 20 hours. The scale-up capability of the method is demonstrated with 2 liter fermenting mixture. The method does not consume external energy due to its electrostatic nature. Our results indicate the applicability of the fermentation technique to industry applications.

Controlled glucose consumption in yeast using a transistor-like device.

From the point of view of systems biology, insight into controlling the functioning of biological systems is conducive to the understanding of their complexness. The development of novel devices, instrumentation and approaches facilitates this endeavor. Here, we show a transistor-like device that can be used to control the kinetics of the consumption of glucose at a yeast-immobilised electrode. The gating voltage of the device applied at an insulated gating electrode was used to control both the rate of glucose consumption and the rate of the production of ATP and ethanol, the end-products of normal glucose metabolism. Further, a correlation between the glucose consumption and the production of ethanol controlled by the gating voltage was observed using two different forms of the device. The results suggest the relevance of glucose metabolism in our work and demonstrate the electrostatic nature of the device. An attempt to explain the effect of the gating voltage on the kinetics is made in terms of transfer of electrons from NADH to enzymes in the electron transport chain. This novel technique is applicable to general cells and the reported results show a possible role for electrostatic means in controlling processes in cells.

Voltage-controlled enzyme-catalyzed glucose-gluconolactone conversion using a field-effect enzymatic detector.

The field-effect enzymatic detection (FEED) technique was used to control the kinetics of the enzymatic conversion of glucose to gluconolactone. The glucose-gluconolactone conversion occurring at an enzyme-immobilized electrode, a well-studied process, was confirmed using mass spectrometry. Electrochemical studies showed that the glucose oxidation current depends on the gating voltage VG and the ion concentration of the sample solution. Additionally, the depletion of glucose in the sample also showed a dependence on VG. FEED was used to detect H2O2 on the zepto-molar level in order to show the ultrasensitive detection capability of the technique. These results, while providing evidence for the proposed mechanism of FEED, indicate that VG controls the conversion process. The effect of VG on the glucose-gluconolactone conversion was demonstrated by the observed VG-dependent kinetic parameters of the conversion process.

Field-effect amperometric immuno-detection of protein biomarker.

The field-effect enzymatic detection technique has been applied to the amperometric immunoassay of the cancer biomarker, carcinoma antigen 125 (CA 125). The detection adopted a reagentless approach, in which the analyte, CA 125, was immobilized on the detecting electrode, which was modified using carbon nanotubes, and the detection signal was obtained by measuring the reduction peak current of the enzyme that was used to label the antibody. A gating voltage was applied to the detecting electrode, inducing increase in the signal current and therefore providing amplification of the detection signal. The voltage-controlled signal amplification of the detection system has increased the sensitivity and lowered the detection limit of the system. A detection limit of 0.9U/ml was obtained in the work.

Ultrasensitive biosensing on the zepto-molar level.

Detection of analytes on the zepto-molar (10(-21) M) level has been achieved using a field-effect bio-detector. By applying a gating voltage to enzymes immobilized on the working electrode of the detector, amplification of the biocatalytic current was observed. The amplification is attributed to the modification of the tunnel barrier between the enzyme and the electrode by the gating voltage-induced electric field which exists at the solution-electrode interface. The detection was demonstrated with the glucose oxidase (GOx)-glucose and alcohol dehydrogenase (ADH)-ethanol biocatalytic systems. Glucose at zepto-molar level was detected with zepto-molar detection resolution. Equivalently, 30 glucose molecules present in the sample were detected and the detection system responded distinctively to the incremental change in the number of glucose molecules in unit of 30 molecules. The enzyme's biospecificity was also preserved in the presence of the applied field. We present possible processes that could give rise to the electrical charges required to produce the observed current level.

Field-effect enzymatic amplifying detector with picomolar detection limit.

Voltage-controlled amplification of the output current of an enzymatic detector has been demonstrated. By application of an external voltage between the gating electrode and the working electrode on which the enzyme glucose oxidase was immobilized, the biocatalytic output current of the detector was increased significantly, allowing the detection limit of glucose to be lowered from the millimolar level to the picomolar level. The current amplification could be reversibly controlled by the applied voltage. Application of this technique to the ethanol-alcohol dehydrogenase system showed similar results. The detection setup suggests that the output current is controlled by the electric field at the interface between the solution and the working electrode. The enzyme's biospecificity was preserved in the presence of the field. The detector, with its output current controlled by a voltage applied at a third electrode, behaves as a field-effect transistor, whose current-generating mechanism is the conversion of an analyte to a product using an enzyme as catalyst. In a broader sense, the operation of the detector shows a means for manipulating a redox enzymatic reaction.

A hybrid biofuel cell based on electrooxidation of glucose using ultra-small silicon nanoparticles.

The ultra-small silicon nanoparticle was shown to be an electrocatalyst for the electrooxidation of glucose. The oxidation appeared to be a first order reaction which involves the transfer of 1 electron. The oxidation potential showed a low onset of -0.4V vs. Ag/AgCl (-0.62 V vs. RHE). The particle was used as the anode catalyst of a prototype hybrid biofuel cell, which operated on glucose and hydrogen peroxide. The output power of the hybrid cell showed a dependence on the enzymes used as the cathode catalyst. The power density was optimized to 3.7 microW/cm(2) when horseradish peroxidase was replaced by microperoxidase-11 (MP-11). Comparing the output power of the hybrid cell to that of a biofuel cell indicates enhanced cell performance due to the fast reaction kinetics of the particle. The long-term stability of the hybrid cell was characterized by monitoring the cell voltage for 5 days. It appeared to that the robustness of the silicon particle resulted in more cell stability compared to the long-term performance of a biofuel cell.

Preserved enzymatic activity of glucose oxidase immobilized on unmodified electrodes for glucose detection.

Glucose sensing electrodes have been realized by immobilizing glucose oxidase (GOx) on unmodified edge plane of highly oriented pyrolytic graphite (epHOPG) and the native oxide of heavily doped silicon (SiO2/Si). Both kinds of electrode show direct interfacial electron transfer due to the redox process of the immobilized GOx. The measured formal potential of the redox process agrees with that of the native enzyme, suggesting that the immobilized GOx has retained its enzymatic activity. The electron transfer rates of the GOx immobilized electrode are 2s(-1) for GOx/epHOPG electrode and 7.9s(-1) for GOx/SiO2/Si electrode, which are greater than those for which GOx is immobilized on modified electrodes, probably due to the fact that the enzyme makes direct contact to electrode surface. The preservation of the enzymatic activity of the immobilized GOx has been confirmed by observing the response of the GOx/epHOPG and GOx/SiO2/Si electrodes to glucose with a detection limit of 0.050 mM. The response signals the catalyzed oxidation of glucose and, therefore, confirms that the immobilized GOx retained its enzymatic activity. The properties of the electrode as a glucose sensor are presented.

Inlaying nanoscale surface recess patterns with nanoscale objects.

A simple and versatile approach to constructing patterns on a solid surface using nanoscale objects is demonstrated. The approach is essentially an inlaying process, in which recess patterns fabricated on a surface are selectively filled with nanoscale objects. The objects are anchored firmly on the surface due to the spatial confinement provided by the recess structures. Protein molecules and inorganic nanoparticles are used in this demonstration. Cyclic voltammetry is used to detect electron transfer signals from patterns of protein molecules. The approach suggests a potentially fast, high-throughput and versatile technique for constructing architectural structures on a solid surface using nanoscale objects.

Enhancing electron transfer at a cytochrome c-immobilized microelectrode and macroelectrode.

The redox reaction of cytochrome c immobilized on the bare surfaces of microelectrodes and macroscopic electrodes (macroelectrodes) composed of different planes of highly oriented pyrolytic graphite has been investigated using cyclic voltammetry. The protein-immobilized microelectrodes were fabricated using a simple masking method. For both macroelectrodes and microelectrodes, the redox reaction of immobilized cytochrome c needs to be activated by increasing the electrochemical potential maximum of cyclic voltammetry to a high positive value. The redox currents of this protein-electrode system can be enhanced using two approaches. The oxidation and reduction currents of cytochrome c adsorbed on microelectrodes that are composed of the edge plane show an anomalous enhancement compared to those for macroelectrodes composed of the basal plane. The difference in the surface chemical properties of the two kinds of electrodes results in the current anomaly. The oxidation current of the macroelectrode can be selectively enhanced by decreasing the potential minimum.

Solvent entropy contribution to the free energy of protein crystallization.

We show with three proteins that trapping and release of the water molecules upon crystallization is a determinant of the crystallization thermodynamics. With HbC, a strong retrograde solubility dependence on temperature yields a high positive enthalpy of 155 kJ mol(-1), i.e., crystallization is only possible because of the huge entropy gain of 610 J mol(-1) x K(-1), stemming from the release of up to 10 water molecules per protein intermolecular contact. With apoferritin, the enthalpy of crystallization is close to zero. The main component in the crystallization driving force is the entropy gain due to the release upon crystallization of two water molecules bound to one protein molecules in solution. With both proteins, the density of the growth sites imaged by AFM is in excellent agreement with a calculation using the crystallization free energy. With lysozyme, the entropy effect due to the restructuring of the water molecules is negative. This leads to higher solubility.