Small Sample Volumes Research Articles

In-fiber optofluidic michelson interferometer for detecting small volume and low concentration chemicals with a fiber ring cavity laser

An in-fiber optofluidic Michelson interferometer fabricated by femtosecond laser micromachining technology was presented. The interferometer structure was prepared by a special optical fiber with two cores and a microchannel. One core was connected to the core of a single-mode fiber (SMF) and functioned as a sensing arm on the microchannel inner wall, the other core worked as a reference arm and was connected to the optical waveguide introduced by femtosecond laser processing. By combining with a ring cavity laser, the detection limit (DL) of the interferometer could be improved. The laser sensor presented a high linear response with a refractive index (RI) sensitivity of 1039.77 nm/RIU and the calculated DL could reach as low as 1.675 × 10 −5 RIU. Then, the sensor measured different concentrations of vitamin C in tablets in the concentration range of 0–10 mg/mL with a detection limit of 0.06 mg/mL. Due to the advantages of optofluidic structure, the volume of the detection solution could be as low as few tens of nanoliters, and the time to replace and detect the solution only took a few seconds. In addition, the laser sensor realized label-free DNA hybridization detection through surface functionalization, and could detect 1 μM target DNA solution with high specificity. This sensing device had great potential for small volume and low concentration sample detection in the biochemical field. • An in-fiber optofluidic Michelson interferometer for detecting small volume and low concentration chemicals is presented. • The interferometer structure is prepared by a special microstructured optical fiber with two cores and a microchannel. • One core is connected with the SMF core as a sensing arm on the microchannel inner wall. • The other core is connected with an optical waveguide from the SMF core as a reference arm in the cladding. • Detection limit can reach as low as 0.06 mg/mL.

SARS-CoV-2 severity classification from plasma sample using confocal Raman spectroscopy.

The world is on the brink of facing coronavirus's (COVID-19) fourth wave as the mutant forms of viruses are escaping neutralizing antibodies in spite of being vaccinated. As we have already witnessed that it has encumbered our health system, with hospitals swamped with infected patients observed during the viral outbreak. Rapid triage of patients infected with SARS-CoV-2 is required during hospitalization to prioritize and provide the best point of care. Traditional diagnostics techniques such as RT-PCR and clinical parameters such as symptoms, comorbidities, sex and age are not enough to identify the severity of patients. Here, we investigated the potential of confocal Raman microspectroscopy as a powerful tool to generate an expeditious blood-based test for the classification of COVID-19 disease severity using 65 patients plasma samples from cohorts infected with SARS-CoV-2. We designed an easy manageable blood test where we used a small volume (8μl) of inactivated whole plasma samples from infected patients without any extra solvent usage in plasma processing. Raman spectra of plasma samples were acquired and multivariate exploratory analysis PC-LDA (principal component based linear discriminant analysis) was used to build a model, which segregated the severe from the non-severe COVID-19 group with a sensitivity of 83.87%, specificity of 70.60% and classification efficiency of 76.92%. Among the bands expressed in both the cohorts, the study led to the identification of Raman fingerprint regions corresponding to lipids (1661, 1742), proteins amide I and amide III (1555, 1247), proteins (Phe) (1006, 1034), and nucleic acids (760) to be differentially expressed in severe COVID-19 patient's samples. In summary, the current study exhibits the potential of confocal Raman to generate simple, rapid, and less expensive blood tests to triage the severity of patients infected with SARS-CoV-2.

Rheological and interface adhesive properties of osteoarthritic synovial fluids

Lubrication by synovial fluid is vital in maintaining the physiological operation of diarthrodial joints, which can be compromised due to changes in synovial fluid as osteoarthritis progresses, resulting in a lubrication deficiency. We studied the rheological and interface adhesive properties of osteoarthritic synovial fluid samples (49 in total), aiming to establish the correlation with medical conditions experienced by patients who underwent knee or hip Total Joint Replacement due to osteoarthritis. The steady-state rheology and the interfacial adhesion energy of those samples were examined, of which the results were compared across various patient characteristics. Seniority (60–79 years old) and obesity (Body Mass Index above 30) were identified as the dominant risk factors for the degeneration of synovial fluid and, thus, the potential development of osteoarthritis. At low shear rates where the interactions are more prominent than high shear rates, synovial fluid viscosity decreased by 58% with age and 38% with Body Mass Index, while the shear-thinning index increased by 40% and 7%, respectively. Additionally, synovial fluid interfacial adhesion properties deteriorate, as shown by the adhesion energy results. The adhesion energy, which is positively correlated to the coefficient of friction, increased by 172% with age and by 234% with Body Mass Index. The changes in both rheological and interface adhesion properties of synovial fluid samples support the relationship between the physico-chemical properties and the clinical observation. The AFM based surface adhesion method could be considered as a new approach to evaluate the tribological characteristics of natural synovial fluids as it requires small volume of samples.

Investigation of Attitudes and Behaviors Towards Recycling with Theory Planned Behavior

Recycling is defined as the re-inclusion of wastes that can be reused in the production process by undergoing various processes. At the same time, recycling is seen as the most important environmental behavior that reduces the amount of solid waste and conserves resources. Considering the importance of recycling for a sustainable future, it is of great importance to determine the factors affecting the recycling behavior of individuals. Due to the rapid increase in environmental problems and their extremely negative impact on life, it is necessary to determine the environmental and recycling behaviors of individuals and produce solutions. In this study, attitudes and behaviors towards recycling were investigated with the help of a model proposed within the scope of Theory Planned Behavior (TPB). The analysis of the data was made using the Partial Least Squares Structural Equation Modeling (PLSSEM) Smart-PLS software, which can be used successfully in small-volume samples and does not require the assumption of multivariate normality. It was determined because of PLS-SEM fit criteria that TPB is suitable for explaining recycling attitudes and behaviors. As a result of the analysis, it was revealed that attitude, subjective norm, and perceived behavioral control positively and significantly affect recycling intentions.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

An automated online three-phase electro-extraction setup with machine-vision process monitoring hyphenated to LC-MS analysis

Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i.e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL−1) analytes and a low starting sample volume of 20 μL of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows.

Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry of 5-Aminolevulinic Acid for Low Concentration and Small Sample Volumes

Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry of 5-Aminolevulinic Acid for Low Concentration and Small Sample Volumes

Wettability-based ultrasensitive detection of amphiphiles through directed concentration at disordered regions in self-assembled monolayers

Various forms of ecological monitoring and disease diagnosis rely upon the detection of amphiphiles, including lipids, lipopolysaccharides, and lipoproteins, at ultralow concentrations in small droplets. Although assays based on droplets' wettability provide promising options in some cases, their reliance on the measurements of surface and bulk properties of whole droplets (e.g., contact angles, surface tensions) makes it difficult to monitor trace amounts of these amphiphiles within small-volume samples. Here, we report a design principle in which self-assembled monolayer-functionalized microstructured surfaces coated with silicone oil create locally disordered regions within a droplet's contact lines to effectively concentrate amphiphiles within the areas that dominate the droplet static friction. Remarkably, such surfaces enable the ultrasensitive, naked-eye detection of amphiphiles through changes in the droplets' sliding angles, even when the concentration is four to five orders of magnitude below their critical micelle concentration. We develop a thermodynamic model to explain the partitioning of amphiphiles at the contact line by their cooperative association within the disordered, loosely packed regions of the self-assembled monolayer. Based on this local analyte concentrating effect, we showcase laboratory-on-a-chip surfaces with positionally dependent pinning forces capable of both detecting industrially and biologically relevant amphiphiles (e.g., bacterial endotoxins), as well as sorting aqueous droplets into discrete groups based on their amphiphile concentrations. Furthermore, we demonstrate that the sliding behavior of amphiphile-laden aqueous droplets provides insight into the amphiphile's effective length, thereby allowing these surfaces to discriminate between analytes with highly disparate molecular sizes.

A highly sensitive, easy-and-rapidly-fabricable microfluidic electrochemical cell with an enhanced three-dimensional electric field

An NP-μFEC is a reusable, novel microfluidic electrochemical cell with multiple non-planar interdigitated microelectrode arrays, minimal sample volume, and enhanced electric field penetration for highly sensitive electrochemical analysis. (i) The NP-μFEC features spatial 3-electrode architecture, and a small sample volume (∼4 μL). (ii) Here, [Fe(CN)6]3-/4- redox couple are used as an electrochemical reporter. The effects on the electrochemical properties of NP-μFEC due to the change in the reference electrode (RE) and counter electrode (CE)'s position with respect to the working electrode (WE) position are analyzed. For NP-μFEC, the position of the RE with respect to the WE does not affect the CV, DPV electrochemical profiles. However, the spacing between the CE and WE plays a significant role. (iii) The enhanced three-dimensional electric field penetration in NP-μFEC is validated by finite element analysis simulation using COMSOL Multiphysics. (iv) Without electrode surface modifications, NP-μFEC shows a detection limit (DL) of ∼2.54 × 10−6 M for aqueous [Fe(CN)6]3-/4- probe. (v) The DL for Cu2+, Fe3+, and Hg2+ are 30.5±9.5 μg L−1, 181±58.5 μg L−1, and 12.4±1.95 μg L−1, respectively, which meets the US Environmental Protection Agency (EPA)'s water contamination level for Cu, Fe, and is close to that for Hg (EPA limits are 1300 μg L−1, 300 μg L−1, and 2 μg L−1, respectively). (vi) Further, using a pressure-sensitive adhesive layer to form the channel and create the NP-μFEC configuration simplifies the manufacturing process, making it cost-effective and allowing for rapid adoption in any research lab. NP-μFEC is used to detect heavy metal ions in water. This demonstrates that cost-effective, easy-to-fabricate NP-μFEC can be a new sensitive electrochemical platform.

Bacterial concentration and detection using an ultrasonic nanosieve within a microfluidic device

Standard methods, such as plate counting, to detect bacteria in samples where only small volumes and low concentrations are available, will result in a negative detection, unless additional enriching steps, such as culturing, are used. However, these are laborious and time consuming, which may prevent their effective application to time sensitive situations, for example in clinical settings or for food quality control. Microfluidic concentration of bacterial cells can address this issue, enabling accurate detection and quantification in low abundance samples even when only small sample volumes are used. In this work we use a packed bed of microparticles trapped in a microfluidic chip, that are activated with surface acoustic waves to periodically concentrate and detect bacteria from sample volumes below 10 µL. We demonstrate a bacterial capturing efficiency of 99% and further demonstrate that the concentrated bacteria can be recovered with an 80% efficiency. This highly concentrated recovered sample can then be successfully used in standard methods, such as plate counting and PCR, for the detection of the bacteria using just 1 µL of sample without the need for a culture-based enrichment process. When integrating our ultrasonic nanosieve with fluorescence sensing, it is possible to achieve rapid detection of a wide range of bacteria concentrations. The device enables the fluorescence detection of bacteria concentration of 4 × 105 CFU/mL in only 15 s and achieved a limit of detection of 3.25 × 102 CFU/mL with just 32 min of ultrasonic actuation, requiring only 10 µL of sample. These results demonstrate that our device offers a scalable, portable, and affordable method for the monitoring of low bacterial concentration using small sample volumes.

Monitoring autochthonous lung tumors induced by somatic CRISPR gene editing in mice using a secreted luciferase

BackgroundIn vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies.MethodsTo facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes.ResultsWe demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination.ConclusionsOur study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.

Preconcentration and Separation of Gold Nanoparticles from Environmental Waters Using Extraction Techniques Followed by Spectrometric Quantification.

The quantification of gold nanoparticles (AuNP) in environmental samples at ultratrace concentrations can be accurately performed by sophisticated and pricey analytical methods. This paper aims to challenge the analytical potential and advantages of cheaper and equally reliable alternatives that couple the well-established extraction procedures with common spectrometric methods. We discuss several combinations of techniques that are suitable for separation/preconcentration and quantification of AuNP in complex and challenging aqueous matrices, such as tap, river, lake, brook, mineral, and sea waters, as well as wastewaters. Cloud point extraction (CPE) has been successfully combined with electrothermal atomic absorption spectrometry (ETAAS), inductively coupled plasma mass spectrometry (ICP-MS), chemiluminescence (CL), and total reflection X-ray fluorescence spectrometry (TXRF). The major advantage of this approach is the ability to quantify AuNP of different sizes and coatings in a sample with a volume in the order of milliliters. Small volumes of sample (5 mL), dispersive solvent (50 µL), and extraction agent (70 µL) were reported also for surfactant-assisted dispersive liquid–liquid microextraction (SA-DLLME) coupled with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS). The limits of detection (LOD) achieved using different combinations of methods as well as enrichment factors (EF) varied greatly, being 0.004–200 ng L−1 and 8–250, respectively.

Characterization of the Urinary Metagenome and Virome in Healthy Children.

Recent advances in next-generation sequencing and metagenomic studies have provided insights into the microbial profile of different body sites. However, research on the microbial composition of urine is limited, particularly in children. The goal of this study was to optimize and develop reproducible metagenome and virome protocols using a small volume of urine samples collected from healthy children. We collected midstream urine specimens from 40 healthy children. Using the metagenomics shotgun approach, we tested various protocols. Different microbial roots such as Archaea, Bacteria, Eukaryota, and Viruses were successfully identified using our optimized urine protocol. Our data reflected much variation in the microbial fingerprints of children. Girls had significantly higher levels of Firmicutes, whereas boys had significantly higher levels of Actinobacteria. The genus Anaerococcus dominated the urinary bacteriome of healthy girls, with a significant increase in Anaerococcus prevotii, Anaerococcus vaginalis, and Veillonella parvula (p-value < 0.001) when compared with that of boys. An increased relative abundance of Xylanimonas and Arthrobacter, with a significantly high abundance of Arthrobacter sp. FB24 (p-value 0.0028) and Arthrobacter aurescences (p-value 0.015), was observed in boys. The urinary mycobiome showed a significant rise in the genus Malassezia and Malassezia globose fungus (p-value 0.009) in girls, whereas genus Saccharomyces (p-value 0.009) was significantly high in boys. The beta diversity of the urinary mycobiome was found to differ between different age groups. Boys had significantly more Mastadenovirus and Human mastadenovirus-A in their urinary virome than girls. With increasing age, we noticed an increase in the relative abundance of the order Caudovirales. Our optimized protocols allowed us to identify the unique microbes for each sex by using an adequate volume of urine (3–10 mL) to screen for the bacteriome, mycobiome, and virome profiles in the urine of healthy children. To the best of our knowledge, this is the first study to characterize the metagenomics profiles of urine in a healthy pediatric population.

Vanadium-Doped Porous Cobalt Oxide for Its Superior Peroxidase-like Activity in Simultaneous Total Cholesterol and Glucose Determination in Whole Blood Based on a Simple Two-Dimensional Paper-Based Analytical Device.

Vanadium-doped porous Co3O4 (V-porous Co3O4) was synthesized via a simple soft-templating method and used as a superior peroxidase mimic for the simultaneous colorimetric determination of glucose and total cholesterol (TC) in whole blood samples on a two-dimensional microfluidic paper-based analytical device (2D-μPAD). The large surface area and the presence of two metals in V-porous Co3O4 contributed to its excellent catalytic activity toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 3,3',5,5'- tetramethylbenzidine (TMB) with Michaelis-Menten constants (KM) of 0.1301 and 0.0141 mM, respectively. The 2D-μPAD was fabricated using simple wax screen-printing and cutting techniques. The colorimetric reactions of both glucose and TC on 2D-μPAD were simultaneously performed by adding a single drop of a whole blood sample on the sample zone made of the LF1 membrane. After the enzymatic reactions, the generated hydrogen peroxide (H2O2) was oxidized by V-porous Co3O4 to produce hydroxy radicals (•OH), inducing ABTS and TMB to generate colored products. The generated H2O2 was proportional to the intensities of the green and blue products of the glucose and TC systems, respectively. The developed 2D-μPAD required a short analysis time (∼5 min) with small volumes of samples (15 μL of whole blood) whereby no sample preparation was needed. Owing to several advantages including simplicity, low cost, long-term stability, and simultaneous readout, the novel V-porous Co3O4 coupled with 2D-μPAD proved to be promising for practical uses as a pioneering portable device for the determination of glucose, TC, and other important biomarkers without the need of technical supports.

Improving the sensitivity of MAS spheres using a 9.5 mm spherical shell with 219 μL sample volume spinning in a spherical solenoid coil

Spherical rotors in magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments have potential advantages relative to cylindrical rotors in terms of ease of fabrication, low risk of rotor crash, easy sample exchange, and better microwave access. However, one major disadvantage so far of spherical rotors is poor NMR filling factor due to the small sample volume and large cylindrical radiofrequency (RF) coil. Here we present a novel NMR coil geometry in the form of a spherical coil. The spherical coil best fits the spherical sample to maximize sensitivity, while also providing excellent RF homogeneity. We further improve NMR sensitivity by employing a spherical shell as the rotor, thereby maximizing sample volume (219 μL in this case of 9.5 mm outer diameter spheres). The spinning gas is supplied by a 3D-printed ring stator external to the coil, thereby introducing a simplified form of MAS stators. In this apparatus, the RF field generated along the coil axis is perpendicular to the external magnetic field, regardless of rotor orientation. We observe a linear increase in sensitivity with increasing sample volume. We also simulate the RF performance of spherical and cylindrical solenoid coils with constant or variable pitch for spherical and cylindrical rotors, respectively. The simulation results show that spherical solenoid coils generate comparable B1 field intensities but have better homogeneity than cylindrical solenoid coils do.

Primary Vitreoretinal Lymphoma: Current Diagnostic Laboratory Tests and New Emerging Molecular Tools.

Primary vitreoretinal lymphoma (PVRL), a rare aggressive malignancy primarily involving the retina and/or the vitreous, is a major diagnostic challenge for clinicians (who commonly misdiagnose it as chronic uveitis) as well as for pathologists (for biological and technical reasons). Delays in diagnosis and treatment are responsible for visual impairments and life-threatening consequences, usually related to central nervous system involvement. The identification of lymphoma cells in vitreous fluid, obtained by vitrectomy, is required for diagnosis. Of note, the scarcity of neoplastic cells in small volumes of vitreous sample, and the fragility of lymphoma cells with degenerative changes caused by previous steroid use for presumed uveitis makes diagnosis based on cytology plus immunophenotyping difficult. Interleukin levels, immunoglobulin heavy chain or T-cell receptor gene rearrangements, and MYD88 mutation are applied in combination with cytology to support diagnosis. We aim to describe the current laboratory technologies for PVRL diagnosis, focusing on the main issues that these methods have. In addition, new emerging diagnostic strategies, such as next-generation sequencing analysis, are discussed. The genetic profile of PVRL remains largely unexplored. Better knowledge of genetic alterations is critical for precision medicine interventions with target-based treatments of this lymphoma for which no standardised treatment protocol currently exists.

Mechanical Properties Study of Miniature Steel Specimens Based on the Small Punch Test and Simulation Methods.

The small punch test (SPT) can be very convenient to obtain mechanical properties due to its unique advantages from small-volume samples, and has gained wide popularity and appreciation among researchers. In this paper, the SPT test and finite element (FE) simulations were performed for three alloys, and the yield stresses (σYS) and ultimate tensile strengths (σUTS) from the uniaxial tensile test (UTT) were correlated with the yield force (Fy) and maximum force (Fm) of the small punch test (SPT) before and after compliance calibration. Finally, the effect of specimen size on the SPT curves was discussed. The results showed that the deviation between SPT test and FE simulation was due to the loading system stiffness, which was confirmed by the loading system compliance calibration test. The SPT curves before and after calibration have less influence on the empirical correlation results for σUTS, while the correlation results for σYS depend on the method used to determine Fy in the SPT curve. Finally, the simulation results indicated that the effect of specimen size on the force–displacement curve in the SPT is slight. This work also provides a reference for subsequent researchers to conduct empirical correlation studies using different specimen sizes.

Label-free monitoring of proteins in optofluidic hollow-core photonic crystal fibres

The fluorescent detection of proteins without labels or stains, which affect their behaviour and require additional genetic or chemical preparation, has broad applications to biological research. However, standard approaches require large sample volumes or analyse only a small fraction of the sample. Here we use optofluidic hollow-core photonic crystal fibres to detect and quantify sub-microlitre volumes of unmodified bovine serum albumin (BSA) protein down to 100 nM concentrations. The optofluidic fibre’s waveguiding properties are optimised for guidance at the (auto)fluorescence emission wavelength, enabling fluorescence collection from a 10 cm long excitation region, increasing sensitivity. The observed spectra agree with spectra taken from a conventional cuvette-based fluorimeter, corrected for the guidance properties of the fibre. The BSA fluorescence depended linearly on BSA concentration, while only a small hysteresis effect was observed, suggesting limited biofouling of the fibre sensor. Finally, we briefly discuss how this method could be used to study aggregation kinetics. With small sample volumes, the ability to use unlabelled proteins, and continuous flow, the method will be of interest to a broad range of protein-related research.

Optimizing reduced capture antibody conjugation to encoded hydrogel microparticles for enhanced multiplex immunoassays

Multiplex detection of protein biomarkers in biological fluids facilitates high-throughput detection using small-volume samples, thereby enhancing efficacy of diagnostic assays and proteomic studies. Graphically encoded hydrogel microparticles conjugated with capture antibodies have shown great potential in multiplex immunoassays by providing superior sensitivity and specificity, a broad dynamic range, and large encoding capacity. Recently, the process of post-synthesis conjugation of reduced capture antibodies to unreacted acrylate moieties in hydrogel particles has been developed to efficiently prevent the aggregation of capture antibodies inside particles, which occurs when using conventional conjugation methods. This direct conjugation process yielded robust assay performance through homogeneous conjugation of the capture antibodies, and avoided the use of hydrolytically unstable linker additives. However, no research has been conducted to optimize the process of conjugating capture antibodies to the particles. We here present a strategy to optimize capture antibody conjugation based on the finding that excessive addition of capture antibodies during incubation can rather lower the amount of capture antibodies conjugated to the particles for some types of capture antibodies. Based on our optimized capture antibody conjugation process, a singleplex immunoassay for a selected target was conducted. Enhanced sensitivity compared with previous studies was confirmed. We also validated the increased specificity of multiplex detection through our optimization process. We believe that the optimization process presented herein for capture antibody conjugation will advance the field of encoded hydrogel microparticle-based immunoassays.

Rapid Detection of Inflammation-Related Biomarkers Using an Electrochemical Sensor Modified with a PBNC-AuNS-GO-Based Nanocomposite

Inflammation is the immune system response when the healthy tissues of the body are damaged by different factors, such as bacteria, viruses, and toxins. Also, when widespread inflammation happens in the body, it can lead to specific conditions that are a significant cause of hospital morbidity and mortality worldwide, e.g., sepsis. Though many inflammation-related biomarkers can be used for the diagnosis of the clinical syndrome, most of the standard tests have a long turnaround time, high cost, and the need for medically trained personnel. Considering the urgency to develop a faster and multitarget method to prevent the aggravation of inflammatory responses using a small volume of human blood samples, herein is the first part of a study focusing on the design and fabrication of a flexible polymer-based device, with a metal-based nanocomposite, to detect three inflammation-related biomarkers (lactate, C-reactive protein, and interleukin-6) that are also detected in high levels during the different stages of inflammation. The working electrode surface was modified with Prussian blue nanocubes, gold nanostars, and graphene oxide to improve the sensitivity and selectivity, enhancing the surface area, electrical conductivity, and electroactivity. Differential pulse voltammetry measurements were recorded while the serum sample was injected with different target concentrations in the channel. The bioreceptors recognize and bind the targets changing the peak currents based on the concentration in the buffer solution. The results show a linear behavior, with high values (>0.99) for the correlation coefficients, supporting the relationship between the concentration of inflammation-related markers and the electrical current. With the improvement in the device structure to be adapted as a portable reader connected to a smartphone, the proposed method is validated to provide a point-of-care diagnosis of septic shock in the emergency department of hospitals using a small volume of samples.