Tiny Sample Volumes Research Articles

Ultrasensitive Chip-Liquid Chromatography Analytical Tool for Quantification-A Review

Introduction: Chip liquid chromatography (LC) is a form of liquid chromatography (LC) that has been scaled down to suit a microchip for quick, high-throughput analysis with tiny sample volumes and minimal reagent usage. The term also goes by To date, four distinct on-chip LC strategies have been created. These strategies make use of columns that are open-tubular, packed-particle, monolithic, and pillar array.
 Applications: This technique is used to analyze small compounds and medications in a variety of biological materials, as well as for proteomics. High quantities of other endogenous chemicals that may cause interference are present alongside these biological compounds, which are only present in tiny levels. LC miniaturization offers a solution to such problems.
 Advantage: On-a-chip operations like sampling, sample transfer, separation, and identification are carried out. Sample preparation is the most challenging task at the micro level and is an important component of most analytical techniques, particularly in bioanalysis. One of the crucial steps in liquid chromatography is separation. Sensitive detection methods such as laser-induced fluorescence microscopy and mass spectrometry are being used.

Thermal Lens Measurements of Thermal Expansivity in Thermosensitive Polymer Solutions

The weak absorption of a laser beam generates in a fluid an inhomogeneous refractive index profile acting as a negative lens. This self-effect on beam propagation, known as Thermal Lensing (TL), is extensively exploited in sensitive spectroscopic techniques, and in several all-optical methods for the assessment of thermo-optical properties of simple and complex fluids. Using the Lorentz-Lorenz equation, we show that the TL signal is directly proportional to the sample thermal expansivity α, a feature allowing minute density changes to be detected with high sensitivity in a tiny sample volume, using a simple optical scheme. We took advantage of this key result to investigate the compaction of PniPAM microgels occurring around their volume phase transition temperature, and the temperature-driven formation of poloxamer micelles. For both these different kinds of structural transitions, we observed a significant peak in the solute contribution to α, indicating a decrease in the overall solution density-rather counterintuitive evidence that can nevertheless be attributed to the dehydration of the polymer chains. Finally, we compare the novel method we propose with other techniques currently used to obtain specific volume changes.

Direct immersion solid-phase microextraction combined with ambient ionization tandem mass spectrometry to rapidly distinguish pesticides in serum for emergency diagnostics.

Despite the fact that carbamates and organophosphates cause acute poisoning via different mechanisms and require disparate management, they are indistinguishable by current clinical assays. Herein, direct immersion solid-phase microextraction (DI-SPME) plus thermal desorption-electrospray ionization tandem mass spectrometry (TD-ESI/MS/MS) was developed to discern them. Both pesticides spiked in human serum were extracted by SPME and analyzed by TD-ESI/MS/MS. This is a promising emergency care platform as rapid analyses could be done in tiny sample volumes with satisfactory recovery (89.46%-116.32%), precision (covariance <20%), sensitivity (LOD <0.1 μg/mL), turnaround time (<5 minutes), and linearity (R2 = 0.9827-0.9992) within 0.1-100 μg/mL.

The potential of SERS as an AST methodology in clinical settings

Abstract The ability to identify and characterize microorganisms from tiny sample volumes in a rapid and reliable way is the first and crucial step in the diagnostics of microbial infections. Ideal analytical techniques would require minimal and low-cost sample preparation, permit automatic analysis of many serial samples, and allow rapid classification of present microorganisms against a stable database. Current practice, however, is far from this ideal; a typical analytical procedure might require a few days. Delayed laboratory results might lead, for example, to progress/spread of the infection, more serious condition of the patient, even death, prescription of inappropriate antibiotics that could be ineffective against causative agents and may as well contribute to the emerging problem of drug resistance in microorganisms. Several studies confirmed that surface enhanced Raman scattering (SERS) is capable of a rapid identification and discrimination of biological samples including medically relevant bacteria. A typical spectrum contains a wealth of information indicative of the cellular content of nucleic acids, purine bases, proteins, carbohydrates, and lipids. Such a spectrum functions as a cellular ‘fingerprint’ and serves as a sensitive indicator of the physiological state of the cell which in turn enables to differentiate cell types, actual physiological states, nutrient conditions, and phenotype changes. Consequently, the focus of this review is on the SERS spectra of bacteria which result from secreted metabolic substances – the purine bases – which are a common feature in the label-free SERS research related to clinical diagnostics of pathogens. Here is the review of the current status of SERS applications on bacteria. A special attention is given to the efforts of profiling antimicrobial susceptibility at clinically relevant species, which in turn has a great potential for use in routine point-of-care (POC) tests. Thus, early and accurate infection disease management can be provided at the bedside or at remote care centres.

The Investigation of Lipid Components in Serum of Metabolic Syndrome Patients by using ATR-FTIR Spectral Analysis

Metabolic syndrome (MetS) is manifested with a cluster of cardiovascular risk factors, including central obesity, hypertension, insulin resistance, and dyslipidemia. MetS also has increased risk for atrial fibrillation (AF), the most common arrhythmia which can cause embolic stroke and heart failure. Our previous studies have found a critical role of lipids and lipoproteins modifications in the pathogenesis of atrial remodeling, which contributes to MetS patient's vulnerability to AF. The attenuated total reflectance Fourier transform infrared (ATR-FTIR) analyzing system can provide kinetic infrared absorption for serum metabolites in the spectral range of 4000-650 cm−1, with advantages including tiny sample volume (0.5 μL), rapid generation of data, and no consumption of reagents and expensive supplies. By ATR-FTIR spectral analysis, significant differences of peak height ratio (PHR) between non-MetS and MetS were revealed at two absorptions, including 2960 cm−1 (νasCH3) and 2929 cm−1 (νasCH2), 1122 cm−1 (δ(C-O-H)) of glycans and 1078 cm−1 (δ(C-O-H)) of glycans. The increasing peak height of 2929 cm−1, 1122 cm−1, and 1033 cm−1 was shown powerful to discriminate MetS from non-MetS samples. The aforementioned peaks represent lipid species and oligosaccharides, that are demonstrated as the spectral fingerprints. The characteristic absorptions of IR for methyl group and methylene group, that are dominantly attributed from proteins and lipids respectively, are spectral markers for MetS. The PHR of two peaks at 2929 cm−1 (νasCH2) and 2960 cm−1 (νasCH3) was −1.9±1.8, which was 21.6±5.8 in MetS. Besides, 1122 cm−1 (δ(C-O-H))/1078 cm−1 (δ(C-O-H)) kinetic ratio was −26.2±4.7 in non-MetS and 12.2±2.1 in MetS, respectively. Conclusively, the ATR-FTIR spectral analysis of serum sample is rapid and sensitive for the detection of lipid modifications in MetS. The technique is worth development and will be helpful for AF prevention in MetS.

101-LB: Real-Time Monitoring of Cholesterol Levels Using a Microfluidic Device

The lack of point-of-care (POC) devices that continuously monitor health is an unmet need. The challenge is not just to create POC devices, but to also go beyond the single time point or discrete measurements. For instance, in a critical in-patient setting, the need to monitor patients’ biochemical signals is important; discrete POC devices are not effective and increase clinical burden. For example, continuous glucose monitors have made it easier for patients to track glucose levels, there are currently no other similar devices either in POC or in an in-patient setting. We had previously reported results in which we carried out, in real-time, continuous measurement of albumin from hepatocytes as a marker of liver function. These measurements were carried out at high sensitivity (∼fg) and temporal resolution (&amp;lt;20s) (10.1007/s10544-017-0244-6). In this abstract, we now report novel POC devices that utilize smart chemistry to carry out continuous, real-time measurement of cholesterol using a tiny sample volume (∼1ul) and achieving detection limit surpassing the conventional assays. The response of hepatocytes to simvastatin was accurately measured by the device, again in real-time. Since the devices are easily customizable, by simply changing the enzymes used in the reactions, we can measure glucose, lactate, and urea. These devices can be groundbreaking in personalized tracking of metabolic health in a POC setting as well as for in-patient situation to assess treatments of ongoing interventions without increasing clinical burden. Disclosure A. Bhushan: None. S.J. Karnik: None. Funding National Institutes of Health (DK095984)

Multiple optical trapping assisted bead-array based fluorescence assay of free and total prostate-specific antigen in serum

Although suspension bead-based assay technology has been widely used owing to its advantages of high-throughput and microvolume detection, its sensitivity is greatly limited because it detects the fluorescence signal emitted by microbeads for a short time in the flowing fluid. In this work, we present the approach for prostate-specific antigen (PSA) detection of both free PSA (fPSA) and total PSA (tPSA) based on bead-array based fluorescence imaging by combining multiple optical trapping and bead-based bioassays. The polystyrene beads were employed to enrich the targets using the classic sandwich immuno-binding and tagged with fluorescent quantum dots (QDs), and the QDs-tagged beads in suspension were trapped array-by-array using multiple optical tweezers constructed with a diffraction optical element and excited with a 405 nm fiber laser for wide-field fluorescence imaging. The distinctive size information from the image of the trapped beads enabled the sorting of different targets. Moreover, the limits of detection for fPSA and tPSA are 3.8 pg/mL and 2.5 pg/mL respectively with good specificity. More importantly, this strategy was successfully used to detect fPSA and tPSA simultaneously in real serum samples. The high sensitivity, good selectivity, and tiny sample volume make this strategy a promising method for life sciences and clinical applications.

High-throughput on-line multi-detection for refractive index, velocity, size, and concentration measurements of micro-two-phase flow using optical microfibers

We demonstrate that high-throughput on-line multi-detection (refractive index, segment velocity, size, and concentration) with high resolution can be achieved simultaneously for micro-two-phase flow by microfibers in the microfluidic chip. The microfibers were immersed in the fluid channel in perpendicular to the flow direction with direct contact of test samples to increase the space resolution and detection sensitivity. A novel “soft sealing” method has been developed for microfiber installing in microfluidic chip, where the continuous phase fluid is applied to surround the microfiber and protect it from vibrations and chip-wall contact, to stabilize the microfiber signal. The time-domain microfiber signal is directly applied to quantify the refractive index of test samples, and different operation modes of this measuring technique have been systematically analyzed. With only tiny sample volume (∼100 nanoliters), the measurement resolution for refractive index and concentration can reach 2×10−4 and 2×10−3, respectively. For both velocity and size measurements, the maximum deviation between this measuring technique and image analysis method is less than 3%, while the overall measurement uncertainty is no more than 3.4%.

Fine Specificity of B-Cell Tolerance to Blood Group Antigens Following ABO-Incompatible (Aboi) Heart Transplantation

Introduction: Histo-blood group ABH(O) ags are oligosaccharides present as glycolipids or glycoproteins. A, B or H epitopes are carried by 6 different internal carbohydrate backbones (type I-VI), forming antigenically distinct variants, each type differentially expressed in human tissues. In heart, only type II structures are expressed (on vascular endothelium) whereas red cells express type I-IV. Identifying natural antibody (ab) clones specific to these ABH epitopes may be important in the setting of ABOi transplantation. Our objectives were to 1) develop a diagnostic tool to detect abs specific to ABH epitopes on all 6 carbohydrate backbones and 2) characterize abs in volunteers and patients who received ABOi heart transplants as infants. Methods: Chemically synthesized A type I-VI, B type I-VI and H type I-VI were printed onto microarray slides; spot morphology and ags were confirmed by monoclonal abs to ABH ags. After initial optimization, plasma from different blood group individuals and patients were hybridized and bound IgM, IgG and IgA detected with fluorescently-labelled secondary abs. Slides were scanned with a microarray scanner and Mean Fluorescent Intensities (MFI) determined using Imagene software. Results: Subtype-specific IgM, IgG and IgA abs varied substantially amongst individuals with different blood groups as well as the same blood group. In infants who received ABOi transplants, abs to donor blood group ags were not detected against type II subtype (fig 1) compared to same blood type controls (fig 2). Immunohistochemistry studies on cardiac biopsies confirmed that only type II carbohydrates were expressed on endothelium.[Figure 1][Figure 2]Conclusions: This analysis demonstrates that infant recipients of ABOi transplants remain deficient in production of antibodies to A-ag expressed in the cardiac graft, despite detection of antibodies to irrelevant A-ag detected by agglutination assay. Thus the ABO-microarray is valuable for subtype-specific blood group abs relevant to the graft. This tool will allow enhanced safety of ABO-incompatible organ transplantation and improve study of mechanisms of tolerance and/or accommodation in the clinical setting. Microarray technology provides analysis of tiny sample volumes (< 5 ml plasma), and simultaneous characterization of all ab isotypes, invaluable when testing infants.

Fine Specificity of B-Cell Tolerance to Blood Group Antigens Following ABO-Incompatible (Aboi) Heart Transplantation

Introduction: Histo-blood group ABH(O) ags are oligosaccharides present as glycolipids or glycoproteins. A, B or H epitopes are carried by 6 different internal carbohydrate backbones (type I-VI), forming antigenically distinct variants, each type differentially expressed in human tissues. In heart, only type II structures are expressed (on vascular endothelium) whereas red cells express type I-IV. Identifying natural antibody (ab) clones specific to these ABH epitopes may be important in the setting of ABOi transplantation. Our objectives were to 1) develop a diagnostic tool to detect abs specific to ABH epitopes on all 6 carbohydrate backbones and 2) characterize abs in volunteers and patients who received ABOi heart transplants as infants. Methods: Chemically synthesized A type I-VI, B type I-VI and H type I-VI were printed onto microarray slides; spot morphology and ags were confirmed by monoclonal abs to ABH ags. After initial optimization, plasma from different blood group individuals and patients were hybridized and bound IgM, IgG and IgA detected with fluorescently-labelled secondary abs. Slides were scanned with a microarray scanner and Mean Fluorescent Intensities (MFI) determined using Imagene software. Results: Subtype-specific IgM, IgG and IgA abs varied substantially amongst individuals with different blood groups as well as the same blood group. In infants who received ABOi transplants, abs to donor blood group ags were not detected against type II subtype (fig 1) compared to same blood type controls (fig 2). Immunohistochemistry studies on cardiac biopsies confirmed that only type II carbohydrates were expressed on endothelium.[Figure 1][Figure 2]Conclusions: This analysis demonstrates that infant recipients of ABOi transplants remain deficient in production of antibodies to A-ag expressed in the cardiac graft, despite detection of antibodies to irrelevant A-ag detected by agglutination assay. Thus the ABO-microarray is valuable for subtype-specific blood group abs relevant to the graft. This tool will allow enhanced safety of ABO-incompatible organ transplantation and improve study of mechanisms of tolerance and/or accommodation in the clinical setting. Microarray technology provides analysis of tiny sample volumes (< 5 ml plasma), and simultaneous characterization of all ab isotypes, invaluable when testing infants.

New synthetic discoveries via high-pressure solid-state chemistry

Most of the syntheses in solid-state chemistry are performed at constant pressure of ∼1 atm (about 10(5) Pa) by the manipulation of the thermodynamic parameters temperature and composition, leading to a rich variety of compounds. In contrast, the additional variation of pressure has remained virtually unexplored, due to the relatively large costs of maintaining high-pressure conditions and the inevitably tiny sample volumes. In the last two decades, technical advances, developed for studying the properties of minerals, have found access into the preparative solid-state chemistry, opening up tremendously large areas to synthesize new materials.

Terahertz near-field microspectroscopy

Using near-field, terahertz time-domain spectroscopy (THz-TDS), we investigate how the addition of a dielectric material into a subwavelength-diameter, cylindrical waveguide affects its transmission properties. The THz electric near-field is imaged with deep subwavelength resolution as it emerges from filled and unfilled waveguides. Spectroscopic data measured for waveguides filled with polycrystalline D-tartaric acid, and with polyethylene and silicon powders, illustrate the feasibility of this approach for obtaining spectroscopic information from a tiny sample volume.

Gravitation-driven stress-reduced cell handling

We present a simple lab-on-chip device for handling small samples of delicate cells, e.g. stem cells. It uses a combination of sedimentation and dielectrophoresis. The transport of cells is driven by gravitation. Dielectrophoresis uses radio-frequency electric fields for generating particle-selective forces dependent on size and polarisability. Electrodes along the channels hold particles and/or cells in a defined position and deflect them towards different outlets. The absence of external pumping and the integration of injection and sampling ports allow the processing of tiny sample volumes. Various functions are demonstrated, such as contact-free cell trapping and cell/particle sorting. Pairs of human cells and antibody-coated beads, as they are formed for T cell activation, are separated from unbound beads. The cells experience only low stress levels compared with the stress levels in dielectrophoresis systems, where transport depends on external pumping. Our device is a versatile yet simple tool that finds applications in cellular biotechnology, in particular when an economic solution is required.

Diffuse X-ray scattering from tiny sample volumes

Abstract The emergence of intense synchrotron X-ray sources, efficient focusing optics and high-performance X-ray sensitive area detectors allows for measurements of diffuse scattering from cubic micron-scale sample vol umes. Here we present an experiment that illustrates methods for studying the local structure and defect content of tiny sample volumes. In the experiment, an X-ray microbeam illuminating about ∼5 μm3 of a Ni-based superalloy single crystal, is used to collect Laue patterns and reciprocal space volume maps around fundamental and a superstructure reflections. This measurement illustrates how diffuse reciprocal-space distributions can be collected with good spatial and momentum-transfer resolution from a tiny real-space sample volume. This example demonstrates that emerging diffuse scattering techniques can provide fundamentally new information about crystallographic organization and defect content over many length scales.

Confocal Fluorescence Microscopy for High-Throughput Screening of G-Protein Coupled Receptors

In the pharmaceutical industry, G-protein coupled receptors (GPCRs) are the most successful group of therapeutic targets. Finding compounds that interfere with the ligand-GPCR interaction in a specific and selective way is a major focus of pharmaceutical research today. As compound libraries of large pharmaceutical companies have increased to hundreds of thousands of test compounds, there is a growing need for miniaturization of drug discovery assays to save bioreagents and to reduce the consumption of test compounds. Due to its high sensitivity combined with a femtoliter-sized measurement volume, confocal fluorescence microscopy enables designs for GPCR binding assays with tiny sample volumes. The GPCRs are prepared in the form of plasma membrane fragments from GPCR-overexpressing cells or may be integrated into virus-like particles (VLiPs). One technique to extract binding data from confocal fluorescence experiments is the so-called fluorescence intensity distribution analysis (FIDA). In this review article, we describe the applicability of FIDA to GPCR-focussed high-throughput screening (HTS) and compare FIDA to two other GPCR-adaptable drug discovery techniques for ligand binding studies, the scintillation proximity assay (SPA) and macroscopic fluorescence polarization (FP) measurements. FIDA measures the absolute concentrations of both GPCR-bound and unbound ligand, thereby providing an internal control to the drug screening data. FIDA is amenable to work with relatively low amounts of GPCRs so that the assay may be carried out with biomembranes of a low GPCR density. Moreover, the fluorescence intensity readout of the FIDA technique may be combined with other confocal fluorescence readouts such as fluorescence anisotropy or lifetime. The combination of a low sample volume with an information-rich measurement means that confocal fluorescence spectroscopy can bring substantial benefits as a bioassay platform to pharmaceutical GPCR-directed research.

Diffuse X-ray scattering from tiny sample volumes

Diffuse X-ray scattering from tiny sample volumes