Undiluted Samples Research Articles

Electrochemically activated copper nitroprusside as a catalyst for sensing of carcinogenic acetaldehyde in red wine

Acetaldehyde (AcH) is a carcinogenic, neurotoxic and genotoxic volatile organic compound present in beverages and pose hazardous effects on human health. Herein, we report electrochemically activated copper nitroprusside (CuNPr)-based sensor for the ultra-trace detection of AcH. CuNPr was synthesized by simple co-precipitation method and its physicochemical properties were characterized using spectroscopy and microscopy techniques. CuNPr-modified glassy carbon electrode (CuNPr/GCE) has been electrochemically activated prior to deploying for AcH sensing. The influence of electrochemical activation and supporting electrolytes (LiCl, NaCl, KCl, CsCl, KNO3, Na2HPO4/NaH2PO4, K2HPO4/KH2PO4) have been investigated. A better sensitivity of the sensor was obtained with potassium phosphate (K2HPO4/KH2PO4, pH 7.4) buffer. The electrochemical oxidation of AcH to acetate ions by CuNPr has been studied using in-situ spectroelectrochemical (UV absorption and Raman spectroscopy) analyses. Under optimized differential pulse voltammetric conditions, electrochemically activated CuNPr/GCE exhibited a wide linear range (2.5 × 10−8 – 2.5 × 10−1 M) with a limit of detection towards AcH as low as 41 × 10−8 M. The developed CuNPr/GCE sensor was employed for the sensing of AcH in undiluted commercial red wine sample and found to have good stability and anti-interference ability towards various co-existing ions in undiluted red wine.

Click reaction-assisted construction of antifouling immunosensors for electrochemical detection of cancer biomarkers in human serum

Herein, an electrochemical immunosensor capable of detecting targets in human serum with ultralow fouling and high sensitivity was successfully constructed through a click reaction. One designed biotinylated peptide was associated with the other peptide functionalized with biotin and azide groups at its two terminals, to form a branched antifouling peptide through the streptavidin-biotin affinity interaction, and it was attached to the electrode to construct an antifouling interface. Then, the exposed azide group on the branched peptide was used to link the antibody modified with 5′-dibenzocyclooctyne (DBCO), and the immunosensor was thus constructed with the aid of the click reaction between the azide group and the DBCO. The developed antifouling immunosensor demonstrated a wide linear response range for carcinoembryonic antigen (CEA) and a low limit of detection of 40 fg mL−1, which is much superior to the sensing performance of the immunosensor prepared through the normal EDC/NHS method. The immunosensor was capable of assaying CEA in undiluted serum samples with satisfying accuracy when compared with a clinically approved method. This work offered an effective strategy to develop various biosensors through the conjugation of antifouling peptides with different antibodies.

Interleukin-29 profiles in COVID-19 patients: Survival is associated with IL-29 levels.

Interleukin-29 profiles in COVID-19 patients: Survival is associated with IL-29 levels.

Lab-on-paper aptasensor for label-free picomolar detection of a pancreatic hormone in serum

Pancreatic hormones such as insulin play the crucial life-supporting role of glucose metabolism. Due to rapidly growing diabetic disorders and associated health complications globally, combined with the pathogenic viral infections severely affecting people with pre-existing conditions, new user-friendly, affordable molecular diagnostic approaches that offer simplicity, cost-effectiveness, and an ultra-sensitive detection in human biofluids are necessary to improve the global health. In this regard, a low-cost paper device that enables easy monitoring of fasting blood-relevant ultra-low picomolar insulin levels is innovative, giving broader applications to any other similar critical molecular targets. The essential contribution of this lab-on-paper is the combination of label-free electrochemical insulin aptasensing with a paper electrode device as a simpler, cheaper, and reliable molecular diagnostic approach for complex serum samples with supporting independent validation methods for confirming scientific rigor and fit-for-purpose of the presented approach. Our aptasensor features a carboxylated graphene-aptamer surface design offering a clinically relevant quantitative detection of picomolar insulin present in a 10-fold diluted neat serum based on interfacial capacitance changes in proportion to the serum insulin concentration (dynamic range 5–500 pM and limit of detection 1.5 pM). Application for undiluted serum samples is demonstrated. Furthermore, analysis of an actual diabetes patient serum sample and correlation of the capacitance sensor results with peroxidase antibody label-based insulin assay approaches are presented (amperometric detection and a commercial enzyme-linked immunosorbent assay are used as validation methods).

Reliability of Biodegradation Measurements for Inhibitive Industrial Wastewaters

Industrial wastewaters may contain toxic or highly inhibitive compounds, which makes the measurement of biological oxygen demand (BOD) challenging. Due to the high concentration of organic compounds within them, industrial wastewater samples must be diluted to perform BOD measurements. This study focused on determining the reliability of wastewater BOD measurement using two different types of industrial wastewater, namely pharmaceutical wastewater containing a total organic carbon (TOC) value of 34,000 mg(C)/L and industrial paper manufacturing wastewater containing a corresponding TOC value of 30 mg(C)/L. Both manometric respirometry and the closed-bottle method were used in the study, and the results were compared. It was found that the dilution wastewaters containing inhibitive compounds affected BOD values, which increased due to the decreased inhibiting effect of wastewater pollutants. Therefore, the correct BOD for effluents should be measured from undiluted samples, while the diluted value is appropriate for determining the maximum value for biodegradable organic material in the effluent. The accuracy of the results from the blank samples was also examined, and it was found that the readings of these were different to those from the samples. Therefore, the blank value that must be subtracted may differ depending on the sample.

Phenolics-Linked Antioxidant and Anti-hyperglycemic Properties of Edible Roselle (Hibiscus sabdariffa Linn.) Calyces Targeting Type 2 Diabetes Nutraceutical Benefits in vitro

Phenolics- enriched plant food sources are excellent dietary and therapeutic targets to combat the increasing prevalence of diet and lifestyle-influenced non-communicable chronic diseases (NCDs), such as type 2 diabetes (T2D). Among plant sources, edible flowers rich in health protective phenolic compounds provide novel opportunities as ingredient and nutraceutical sources. Roselle (Hibiscus sabdariffa Linn.) is a popular edible flower and consumed as part of traditional cuisines and processed foods in several countries of Asia and Africa. Red calyces of Roselle are rich in phenolic compounds, which potentially have high antioxidant and anti-hyperglycemic properties. Therefore, there is merit in screening of dried Roselle calyces as sources for functional food ingredients or nutraceuticals to counter chronic oxidative stress and chronic hyperglycemia using in vitro assays. This has led to this study to investigate and compare phenolic compounds associated antioxidant and anti-hyperglycemic functions of different organic solvent-extracted fractions of dried Roselle calyces using rapid in vitro assays-based screening strategy. Total soluble phenolic content, profile of phenolic compounds, free radical scavenging assay-based total antioxidant activity, and anti-hyperglycemic function linked α-amylase and α-glucosidase inhibitory activities of four different organic solvents (chloroform, hexane, ethyl acetate, and initial crude extraction in 100% methanol) extracted fractions of calyces of Roselle were determined using in vitro assays. Studies indicated high phenolic-linked antioxidant and anti-hyperglycemic relevant properties in red Roselle calyces, specifically in ethyl acetate and methanol solvent-based extracted fractions. Major phenolic compounds in extracted fractions of Roselle calyces were chlorogenic acid, caffeic acid, gallic acid, catechin, rutin, benzoic acid, and cinnamic acid. Additionally, moderate α-amylase (30–92%) and very high α-glucosidase (81–98%) inhibitory activities were confirmed in undiluted samples of organic solvent-extracted fractions of Roselle calyces in the in vitro assays. Taken together these in vitro screening results indicated that calyces of Roselle are excellent sources of health protective phenolic compounds with high antioxidant and anti-hyperglycemic functions and organic solvent (ethyl acetate and methanol) extracted fractions of this edible flower can be strategically utilized to design functional food ingredients and nutraceuticals.

Biological sample-compatible Au nanoparticle-containing fluorescent molecularly imprinted polymer microspheres by combining RAFT polymerization and Au-thiol chemistry.

The development of biological sample-compatible fluorescent molecularly imprinted polymers (MIPs) with more functions and/or improved performance is of great importance for various bioanalytical and biomedical applications, but remains challenging. Herein, we report on a versatile strategy for preparing well-defined hydrophilic gold (Au) nanoparticle (AuNP)-containing fluorescent MIP microspheres capable of directly optosensing folic acid (FA) in undiluted urine samples and study of the effect of the incorporated AuNPs on their optosensing sensitivity. Such advanced functional fluorescent MIP particles were readily prepared by combining RAFT polymerization (including RAFT precipitation polymerization and surface-initiated RAFT polymerization) and Au-thiol chemistry [including successive attachment of AuNPs and a thiol-terminated poly(2-hydroxyethyl methacrylate) onto MIP particles after aminolysis of their surface dithioester into thiol groups]. They showed "turn-on" fluorescence and high optosensing selectivity and sensitivity toward FA in the undiluted urine sample (detection limit = 0.088 μM). They also exhibited outstanding photostability and reusability and could directly quantify FA in another undiluted urine sample with good recoveries (96.3-101.6%) and high accuracy (RSD = 0.6-3.1%), even in the presence of several interferents. Unfortunately, the incorporated AuNPs did not improve the optosensing sensitivity of AuNP-containing fluorescent MIPs. Nevertheless, introducing AuNPs onto the surfaces of fluorescent MIPs not only provides an effective new way for grafting with hydrophilic polymer brushes, but it also can endow them with certain new functions [e.g., surface-enhanced Raman scattering (SERS)], thus making them highly promising as both efficient optosensors and potential SERS sensors for rapid FA detection in applications such as clinical diagnostics and food analyses.

Short-term sperm storage protocol for an endangered Mexican fish acúmara (Algansea lacustris)

Cold sperm storage is a simple and inexpensive technique to assist reproduction programs for endangered species. This study aimed to describe sperm quality indicators and develop a cold sperm storage protocol for Algansea lacustris . A total of 8 storage conditions were evaluated: Control 1 (undiluted sperm), Control 2 (sperm diluted in extender BTS™), and 6 different combinations of the extender (Control 2) +: (1) 2.5% Me 2 SO, (2) 5% Me 2 SO, (3) 7.5% Me 2 SO, (4) 10% Me 2 SO, (5) 12.5% Me 2 SO, and (6) 15% Me 2 SO for 0, 24, 48, 72, and 96 hours. The motility and duration of moving sperm were assessed daily for each treatment. Samples diluted in the highest concentrations of Me 2 SO (12.5% and 15%) showed a lower motility rate (60-79%) immediately after dilution (0 h). Undiluted sperm samples (Control 1) showed a higher motility rate (62%) after 72 h and higher duration of motility (33.8 s) after 48 h. The optimal storage condition for obtaining about 60% motility is storing undiluted sperm for up to 72 hours. However, the combination of extender BTS™ at 5% and cryoprotectant Me 2 SO at 10% present future potential for enhancing the spermatic cryopreservation protocol in Algansea lacustris . • Algansea lacustris sperm must be stored for up to 72 hours undiluted. • BTS™ at 5% can be used as an extender in Algansea lacustris sperm. • Cryoprotectant DMSO at 10% has great potential for cryopreservation.

Natural killer cell detection, quantification, and subpopulation identification on paper microfluidic cell chromatography using smartphone-based machine learning classification

Natural killer (NK) cells are immune cells that defend against viral infections and cancer and are used in cancer immunotherapies. Subpopulations of NK cells include CD56dim and CD56bright which either produce cytokines or cytotoxically kill cells directly. The absolute number and proportion of these cells in peripheral blood are tied to proper immune function. Current methods of cytokine detection and proportion of NK cell subpopulations require fluorescent dyes and highly specialized equipment, e.g., flow cytometry, thus rapid cell quantification and subpopulation analysis are needed in the clinical setting. Here, a smartphone-based device and a two-component paper microfluidic chip were used towards identifying NK cell subpopulation and inflammatory markers. One unit measured flow velocity via smartphone-captured video, determining cytokine (IL-2) and total NK cell concentrations in undiluted buffy coat blood samples. The other, single flow lane unit performs spatial separation of CD56dim and CD56bright and cells over its length using differential binding of anti-CD56 nanoparticles. A smartphone microscope combined with cloud-based machine learning predictive modeling (utilizing a random forest classification algorithm) analyzed both flow data and NK cell subpopulation differentiation. Limits of detection for cytokine and cell concentrations were 98 IU/mL and 68 cells/mL, respectively, and cell subpopulation analysis showed 89% accuracy.

Clinical profile, visual outcome and root cause analysis of post-operative cluster endophthalmitis due to Burkholderia cepacia complex.

Purpose:To present varied clinical presentations, surveillance reports, and final visual outcomes of a rare outbreak of cluster endophthalmitis caused by gram-negative, opportunistic bacilli, Burkholderia cepacia complex (Bcc).Methods:Details of five patients who developed postoperative cluster endophthalmitis were collected. For each patient, an undiluted vitreous sample was collected during vitreous tap. Bacterial culture from the vitreous sample in each case had grown Bcc. Surveillance investigations for root cause analysis (RCA) were performed in the operating room (OR), admission, and day-care wards to localize the source.Results:Four patients had undergone phacoemulsification surgery, and one patient had undergone penetrating keratoplasty. Each patient received an initial dose of empiric intravitreal ceftazidime and vancomycin. The organism isolated in each case was sensitive to ceftazidime, cotrimoxazole, and meropenem and resistant to other antibiotics. Core vitrectomy was done after 48–60 hours in four patients along with intravitreal imipenem injection. One patient did not provide consent for core vitrectomy and subsequently developed phthisis bulbi. Three patients had subsequent recurrences. Two patients had a final BCVA of 20/60, two had BCVA better than 20/200, while one patient had no perception of light. None of the surveillance samples from the OR complex could isolate Burkholderia.Conclusion:Extensive OR surveillance should be done to identify the potential source of infection. However, the source may not be identifiable in few instances like in our case. Longer follow-up is recommended in cases of Bcc endophthalmitis due to the persistent nature of the infection.

Determination of Cu and Mn in Seawater by High-Resolution Continuum Source Graphite Furnace Atomic Absorption Spectrometry

The conditions for the determination of copper and manganese in seawater by high-resolution continuum-source graphite furnace atomic absorption spectrometry (HR-CS GFAAS) were optimized. The analytical properties were compared with those of low-resolution line source GFAAS (LR-LS GFAAS). The metrological characteristics were estimated considering the dilution of the sample and optimization of the temperature program of the electrothermal atomizer. Using HR-CS GFAAS, the elements were determined in undiluted samples, while LR-LS GFAAS allows the determination only at double dilution. The lower limits of determined Cu concentrations almost coincided for both versions of GFAAS. For manganese, the limits of detection and quantification were significantly lower for HR-CS GFAAS. Calibration curves were plotted using standard solutions. The proposed scheme was tested on water samples from the Sea of Okhotsk by standard addition. The detection limits (LODs) were 0.6 µg L−1 for copper and 0.07 µg L−1 for manganese by HR-CS GFAAS and 0.5 µg L−1 for copper and 0.13 µg L−1 for manganese by LR-LS GFAAS.

A pretreatment-free electrical capacitance biosensor for exosome detection in undiluted serum

The exosome is considered a useful biomarker for the early diagnosis of cancer. However, pretreatment of samples used in diagnosis is time-consuming. Herein, we fabricated a capacitance-based electrical biosensor that requires no pretreatment of the sample; it is composed of a DNA aptamer/molybdenum disulfide (MoS2) heterolayer on an interdigitated micro-gap electrode (IDMGE)/printed circuit board (PCB) system for detecting exosomes in an undiluted serum sample. The DNA aptamer detects the CD63 protein on the exosome as the biomarker, while the MoS2 nanoparticle enhances electrical sensitivity. In this study, for the first time, the IDMGE system was used to amplify the electrical signal efficiently for exosome detection. The IDMGE amplifies the capacitance signal as the gap between electrodes decreases, making it easy to detect the target by utilizing the heightened sensitivity. Moreover, it is possible to immobilize a bio-probe more efficiently than with an electrical sensitivity–enhancing electrode with the same area. The thiol-modified (SH-) CD63 DNA aptamer was introduced as the bio-probe that selectively binds to the CD63 protein on the exosome surface. The capacitance signal from the IDMGE electrical sensor increased linearly with the increase in the concentration of exosomes in human serum expressed on a logarithmic scale, the detection limit being 2192.6 exosomes/mL. The proposed biosensor can detect exosomes in undiluted human serum with high selectivity and sensitivity. A blind test was also carried out to test the reliability of the biosensor. The capacitance-based electrical biosensor thus offers a new platform for cancer diagnosis in the future.

Analytical performance of Compton/Rayleigh signal ratio by total reflection X‐ray fluorescence (TXRF): A potential methodological tool for sample differentiation

AbstractThe high sensitivity Compton and Rayleigh X‐ray scattering signals can be used to gain valuable information on the chemical composition of various matrices, by exploiting the ratio of those signals as a function of the effective atomic number (Zeff). Neither total reflection X‐ray fluorescence (TXRF) nor the effect of the experimental setup, including sample preparation, X‐ray excitation source selection, and band deconvolution procedure, has been assessed in this kind of approach. Here, a Compton/Rayleigh ratio and Zeff‐based TXRF method was set up and tested as an analytical tool for milk samples differentiation. The method was developed using a 90° scattering angle and assessed using different X‐ray excitation sources: a molybdenum tube (Mo Kα 17.5 KeV) and a tungsten tube (W Lα 8.5 KeV and W‐Brems 35 KeV). The evaluation of independent Compton and Rayleigh signals was performed by non‐Gaussian and Gaussian curve resolution methods, and both height and area‐based calculations were evaluated. Different sample preparation conditions were assessed. By using 11 standard materials, a calibration curve for Compton/Rayleigh ratio versus Zeff was established. The method was tested to determine the Zeff of milk samples, which enabled its use as a parameter to differentiate them. Good precisions were obtained with the Mo excitation source and the area‐based calculations, which allowed to differentiate undiluted milk samples by species, treatment, and fat content according to their Compton/Rayleigh ratio. This simple and rapid method has the potential to be used for the differentiation of various types of samples, including liquids, solids, and aerosols.

Deterministic Cell Separation Recovers >2-Fold T Cells, and More Naïve T Cells, for Autologous Cell Therapy As Compared to Centrifugally Prepared Cells

CAR-T autologous cell therapies are delivering impressive results in the clinic. However, there are still significant manufacturing challenges impeding the rapid adoption of these advanced therapies. On the first day of cell processing, most manufacturing approaches require ~5 steps (~4 hours) to obtain a white blood cell (WBC) preparation sufficiently depleted of red blood cells (RBCs) for T-cell selection and activation steps; and involves large cell losses and a great deal of inconsistency. Here we present a single-step procedure that yields >2 fold more cells that centrifugal processing with comparable or better quality in <1 hour.We previously reported a small-scale microfluidic approach using deterministic cell separation (DCS) to effectively isolate and separate WBCs with high recoveries, no loss of WBC subtypes, no cell damage, and greater numbers of central memory T cells than traditional Ficoll-based processing. Extending this work, we now present the results of our fully scaled-up processing of 23 normal donor leukopaks and 4 disease samples using a full-scale DCS prototype. All samples were processed in <45 minutes, with only an additional 10 minutes hands-on time.On average, inclusive of aggregate removal by prefiltering, DCS achieved 88% WBC recovery, 94% RBC removal, and 98% platelet ( PLT) removal from the undiluted leukopak samples (n=23). Furthermore, DCS resulted in a RBC/WBC ratio of 0.1 compared with a ratio of 1.4 for Ficoll. Similarly, the PLT/WBC ratios were 0.89 versus 7.17 for DCS and Ficoll, respectively (n=20). In addition, DCS preparations contained 2-fold more CD3+ T cells (n=17), and, importantly, the CD4+ cells were less differentiated (more cells in naïve and central memory stages) than those recovered by Ficoll.Similarly, DCS processed blood from cancer patients had a ratio of RBC/WBC = 7.0 versus 20.1 for Ficoll, and a PLT/WBC ratio = 0.7 versus 15.6 for Ficoll (n=4). These results demonstrate the capabilities of DCS in processing not only samples from normal donors but also blood from cancer patients with similar efficiencies.Further, with DCS we achieved wash efficiencies of more than 3 log, without the typically associated cell loss, as demonstrated by the removal of viral particles, soluble proteins and cytokines and growth factors present in plasma. Therefore, cells from leukopaks processed by DCS can be washed and collected directly into cell culture media, or other solutions, to ready them for downstream applications without pelleting and repeated washes, greatly simplifying workflows.We integrated our DCS technology into a full scale parallelized, disposable, closed fluid path solution and automated platform prototype, the Curate ® Cell Processing System, capable of processing undiluted leukopacks at rates in excess of 300mL/hour. Designed to process blood products in bags using a single-use cassette containing microfluidic components, the Curate ® delivers a debulked WBC product to a bag. With a hands-on time of only 10 minutes, the Curate ® reduces the time to activation- and expansion-ready cells from leukopaks by 6-fold as compared with centrifugation and elutriation methods (Bowles, et al. Cytotherapy 2018;20(5):S109). The system can process a full leukopak (200-300 mL containing up to 1.2x10 10 WBC) within 40 minutes with a maximal cell throughput of 1.8x10 10 WBC per hour. Additionally, the same Curate ® device can be used to achieve up to 200x10 6 cell/mL in as little as 40 mL of media and without requiring pelleting.In summary, we believe our technology enables a significant breakthrough in the production of CAR-T cells by efficiently recovering more and cleaner total and naÏve T cells, for CAR-T cell production. Furthermore, the closed-system Curate ® will simplify cell processing workflows by reducing the number of cell washing steps, as well as the hands-on time and resources.Supported in part by NIH Grant No 5R42CA228616-03 DisclosuresBehmardi: GPB Scientific, Inc: Current Employment. Ouaguia: GPB Scientific, Inc: Current Employment. Healey: GPB Scientific, Inc: Current Employment. Jones: GPB Scientific, Inc: Current Employment. Rahmo: GPB Scientific, Inc: Current Employment. Skelley: GPB Scientific, Inc: Current Employment. Gandhi: GPB Scientific, Inc: Current Employment. Campos-Gonzalez: GPB Scientific, Inc: Current Employment, Current holder of stock options in a privately-held company. Civin: GPB Scientific, Inc: Current holder of individual stocks in a privately-held company. Ward: GPB Scientific, Inc: Current Employment.

Erosive potential of commonly available vapes: a cause for concern?

Objectives Relatively little is known about the erosive potential of vape products, an important consideration for dental health. This study analysed flavoured vapes with potentially low pH, including fruit and drink flavours.Methods The pH of 45 purposefully selected vapes was measured undiluted in triplicate. Serial dilution was conducted on the most erosive product to investigate if/when the erosive potential pH of 5.5 was reached. One of the most erosive samples was tested, undiluted and diluted, after four months of opening. Content analysis of the vape labels determined ingredients, place of manufacture, product usage instructions and health warnings.Results Eighty-four percent of undiluted vape samples had a pH below 5.5. Erosive potential was not predicted by the flavour, with orange and cola samples higher than anticipated (pH 3.56-6.23 and 5.04-5.63, respectively). Products that were predicted to be non-acidic such as vanilla showed considerable variation ranging from pH 4.69-5.63. Freshly opened or stored samples did not reach a non-erosive potential pH of 5.5 or above, even when diluted to represent concentrations used when vaped. Labels were not detailed enough to ascertain which ingredients were contributing to acidity or buffering capacity or to allow consumers to identify the least harmful products in terms of oral health.Conclusions Present labelling of commercially available vapes does not allow consumers to ascertain the erosive potential and possible dental damage that may be inflicted by their use. More effective labelling and/or health warnings are required to educate consumers and dental health professionals on these products.

Fluorescent paper strip immunoassay with carbon nanodots@silica for determination of human serum amyloid A1.

A fluorescent paper strip immunoassay in conjunction with carbon nanodots@silica (CND@SiO2) as a label was developed for the quantitative measurements of human serum amyloid A1 (hSAA1) in serum at clinically significant concentrations for lung cancer diagnosis. Monodispersed CND@SiO2 was prepared by cohydrolysis between silane-crosslinked carbon nanodots and silica precursors via the Ströber method and further attached covalently to anti-hSAA1 (14F8) monoclonal antibody [anti-hSAA1(14F8)] specific to the hSAA1 target. The hSAA1 concentrations were then determined by quantifying the blue fluorescence intensity upon 365nm excitation of the captured hSAA1 with anti-hSAA1(14F8)-CND@SiO2 conjugates in the test line on a paper strip where anti-hSAA1 (10G1) monoclonal antibody was physisorbed. The developed fluorescent paper strip with CND@SiO2 can detect hSAA1 at concentrations ranging from 0.1 to 5nM (R2 = 0.995), with a limit of detection of 0.258nM in 10mM phosphate buffer pH 7.4 containing human serum albumin. Theperformance of recovery (90.98-109.17%) and repeatability (coefficients of variation < 8.46%) obtained was also acceptable for quantitative determinations. Theplatform was employed for direct determinationof hSAA1 concentrations in undiluted serum samples from lung cancer patients (relative standard deviation (RSD) < 7.46%) and healthyhumans (RSD < 3.96%). The results were compared with those obtained using a commercially available enzyme-linked immunosorbent assay alongside liquid chromatography with tandem mass spectrometry measurements.

Multiple myeloma: Detection of free monoclonal light chains by modified immunofixation electrophoresis with antisera against free light chains

IntroductionNeoplastic monoclonal gammopathies are frequently associated with production of excess free monoclonal light chains. A sensitive method for detecting free monoclonal light chains in serum could provide a marker for residual/minimal residual disease and as an adjunct to serum protein electrophoresis to serve as a screening method for monoclonal gammopathies. MethodsConventional serum immunofixation electrophoresis was modified by applying undiluted serum samples, and staining for serum free light chains with antisera specific to free light chains. Washing/blotting of gels was enhanced to remove residual proteins that did not bind to the antibodies including intact monoclonal immunoglobulins. Results from this modified immunofixation electrophoresis were compared with results from commercially available MASS-FIX/MALDI assay. ResultsMonoclonal free kappa light chains could be detected to a concentration of about 1.78 mg/L and monoclonal free lambda light chains to a concentration of about 1.15 mg/L without the need for special equipment. Comparison of these thresholds with parallel results from a commercially available MASS-FIX/MALDI assay revealed the modified electrophoretic method to be more sensitive in the context of free monoclonal light chains. ConclusionsModified serum immunofixation electrophoresis has been shown to detect low levels of monoclonal free light chains at a sensitivity suitable for the method to be used in detecting minimal residual disease, and potentially in a screening assay for monoclonal gammopathies. The disparity in the results with commercially available MASS-FIX/MALDI assay is postulated to be due to limited repertoire of reactivity of nanobodies of camelid origin.

Cortisol Detection in Undiluted Human Serum Using a Sensitive Electrochemical Structure-Switching Aptamer over an Antifouling Nanocomposite Layer.

There is a strong and growing need to monitor stress biomarkers in vivo for real-time emotional and wellness assessment. Toward this, we report a reagent-free electrochemical aptasensor with a nanocomposite antifouling layer for sensitive and continuous detection of cortisol in human serum. A thiolated, methylene blue (MB)-tagged conformation-switching aptamer was immobilized over a gold nanowire (AuNW) nanocomposite to capture cortisol and generate a signal proportional to the cortisol concentration. The signal is recorded through differential pulse voltammetry (DPV) and chronoamperometry. The aptasensor exhibited a sensitive response with 0.51 and 0.68 nM detection limits in spiked buffer and undiluted serum samples, respectively. Interference from other structurally similar analogs, namely, epinephrine and cholic acid, was negligible (<10%). The developed nanocomposite-based aptasensor showed excellent stability in undiluted human serum, outperforming several other nanocomposite materials even after prolonged exposure. This work lays the foundation for new biosensor formats such as implantable and wearable sensors.

Lab-on-a-Disc for Point-of-Care Infection Diagnostics

Reliable, inexpensive, and rapid diagnostic tools are essential to control and prevent the spread of infectious diseases. Many commercial kits for coronavirus disease 2019 (COVID-19) diagnostics have played a crucial role in the fight against the COVID-19 pandemic. Most current standard in vitro diagnostic (IVD) protocols for infectious diseases are sensitive but time-consuming and require sophisticated laboratory equipment and specially trained personnel. Recent advances in biosensor technology suggest the potential to deliver point-of-care (POC) diagnostics that are affordable and provide accurate results in a short time. The ideal "sample-in-answer-out" type fully integrated POC infection diagnostic platforms are expected to be autonomous or easy-to-operate, equipment-free or infrastructure-independent, and high-throughput or easy to upscale. In this Account, we detail the recent progress made by our group and others in the development of centrifugal microfluidic devices or lab-on-a-disc (LOAD) systems. Unlike conventional pump-based fluid actuation, the centrifugal force generated by spinning the disc induces liquid pumping and no external fluidic interconnects are required. This allows a total fluidic network required for multiple steps of biological assays to be integrated on a disc, enabling fully automated POC diagnostics. Various applications have been demonstrated, including liquid biopsy for personalized cancer management, food applications, and environmental monitoring; here, we focus on IVD for infectious disease. First, we introduce various on-disc unit operation technologies, including reagent storage, sedimentation, filtration, valving, decanting, aliquoting, mixing, separation, serial dilution, washing, and calibration. Such centrifugal microfluidic technologies have already proved promising for micro-total-analysis systems for automated IVD ranging from molecular detection of pathogens to multiplexed enzyme-linked immunosorbent assays (ELISAs) that use raw samples such as whole blood or saliva. Some recent examples of LOAD systems for molecular diagnostics in which some or all steps of the assays are integrated on a disc, including pathogen enrichment, nucleic acid extraction, amplification, and detection, are discussed in detail. We then introduce fully automated ELISA systems with enhanced sensitivity. Furthermore, we demonstrate a toy-inspired fidget spinner that enables electricity-free and rapid analysis of pathogens from undiluted urine samples of patients with urinary tract infection symptoms and a phenotypic antimicrobial susceptibility test for an extreme POC diagnostics application. Considering the urgent need for cost-effective and reliable POC infection diagnostic tools, especially in the current pandemic crisis, the current limitations and future directions of fast and broad adaptation in real-world settings are also discussed. With proper attention to key challenges and leverage with recent advances in bio-sensing technologies, molecular biology, nanomaterials, analytical chemistry, miniaturization, system integration, and data management, LOAD systems hold the potential to deliver POC infection diagnostic tools with unprecedented performance regarding time, accuracy, and cost. We hope the new insight and promise of LOAD systems for POC infection diagnostics presented in this Account can spark new ideas and inspire further research and development to create better healthcare systems for current and future pandemics.

Femtoamp and picoamp modes of electrospray and paper spray ionization

Sub nanoampere ionization was investigated using glass emitters and paper spray substrates. An electrometer was set up to measure ionization current as low as 20 fA, while the ion trap mass spectrometer was operated to analyze ion current as low as 1 ion per second. Femto amp (100 s fA) ionization currents were generated using glass emitters with 90 nm tip or 4 μm tips. Using the 4 μm emitter tips, both the fA mode and the regular nanoESI mode were generated. Mass spectra produced by the fA mode ESI were >3 order of magnitude less intense than those produced by nanoESI mode. Beyond generating ESI type of ions, fA mode ESI generates native cytochrome c charge states without using any buffering agent. Femto amp ionization was also observed when conducting paper spray ionization, as well as when ionizing dry paper substrate without solvent. Ultra-low current ionization allowed direct analysis of undiluted sample matrices without carryover contamination. Samples collected and stored on a dried paper substrate were analyzed with no sample preparation.