High Analyte Concentrations Research Articles

Combating Prozone Effects and Predicting the Dynamic Range of Naked-Eye Nanoplasmonic Biosensors through Capture Bioentity Optimization.

Accurately quantifying high analyte concentrations poses a challenge due to the common occurrence of the prozone or hook effect within sandwich assays utilized in plasmonic nanoparticle-based lateral flow devices (LFDs). As a result, LFDs are often underestimated compared to other biosensors with concerns surrounding their specificity and sensitivity toward the target analyte. To address this limitation, here we develop an analytical model capable of predicting the prozone effect and subsequently the dynamic range of the biosensor based on the concentration of the capture antibody. To support our model, we conduct a sandwich immunoassay to detect C-reactive protein (CRP) in a phosphate-buffered saline (PBS) buffer using an LFD. Within the experiment, we investigate the relationship between the CRP dynamic range and the prozone effect as a function of the capture antibody concentration, which is increased from 0.1 to 2 mg/mL. The experimental results, while supporting the developed analytical model, show that increasing the capture antibody concentration increases the dynamic range. The developed model therefore holds the potential to expand the measurable range and reduce costs associated with quantifying biomarkers in diverse diagnostic assays. This will ultimately allow LFDs to have better clinical significance before the prozone effect becomes dominant.

Chiroptically active copper clusters as platform for enantioselective detection of lysine.

Metal clusters have drawn considerable research attention over the years due to their fascinating optical properties. Owing to their appealing photophysical characteristics, these materials have drawn attention as potential candidates for various application in diverse fields, including disease detection, biosensing, chemical sensing, and the fabrication of light-harvesting materials. Presently, there is an increasing research focus on the use of clusters in biomedical research, both as biodetection platform and as bioimaging agents. Of special interest are chiral clusters, which can selectively interact with chiral biomolecules owing to their optical activity. Herein, we showcase the use of a pair of chiroptically active copper clusters for the enantioselective detection of lysine, an amino acid of vast biological relevance. Two techniques are concurrently employed for the detection of lysine at varying concentrations. Circular dichroism serves as a potent tool for detecting lysine at low concentrations, whereas luminescence is effectively employed as a detection method for high analyte concentrations. The combined electronic impact of clusters and lysine resulted in the emergence of an enhanced enantioselective Cotton effect at specific wavelength.

Abstract 3667: Nucleic acid quantification in uveal melanoma aqueous humor

Abstract Introduction: Uveal melanoma (UM) is the most common primary adult intraocular cancer. Though radiation brachytherapy is often employed for UM treatment, half of UM patients develop systemic metastases. In large tumors with macular involvement, the disease and its treatment can lead to blindness. Clinically, UM prognosis is estimated by tumor size, location, American Joint Cancer Commission (AJCC) staging, and histologic findings. Prognostic UM biomarkers have been established but lesions <2 mm in height, particularly near the fovea, are difficult to biopsy, and thus physicians often do not perform biopsies on such tumors. Therefore, the use of aqueous humor (AH) as a liquid biopsy is a promising methodology. The purpose of the study was to quantify analytes in AH samples collected at diagnosis and post-brachytherapy from UM eyes with varying tumor sizes and AJCC Stages to analyze correlations between analyte (dsDNA, microDNA, and protein) concentrations and tumor features. Methods: This case series study included 119 UM AH samples from 66 UM eyes and 16 control AH samples collected from age-matched non-UM glaucoma (GLC) eyes analyzed for unprocessed analytes using Qubit fluorescence assays. Results: Most UM AH samples contained quantifiable analyte concentrations (dsDNA: 94.1%, miRNA: 88.0%, protein: 95.2%) and at higher concentrations than GLC controls (dsDNA, P=0.008; miRNA, P<0.0001; protein, P=0.007). In samples taken at diagnosis, concentrations were higher at more advanced AJCC stages; comparing most advanced Stage III (n=8) to least advanced Stage I (n=24), median dsDNA was 4 times greater (P<0.0001), protein was 3 times greater (P<0.0001), and miRNA was 2 times greater (P=0.001). Analytes were quantifiable in >70% of pre-brachytherapy samples from eyes with tumors <2 mm tall (n=17). Height was positively associated with analyte concentrations at diagnosis (dsDNA: R=0.43, P=0.0007; miRNA: R=0.35, P=0.01; protein: R=0.39, P=0.005). In paired samples (n=53), all analyte concentrations were significantly higher post-therapy compared to diagnosis pre-therapy (Ps<0.01). Conclusions: Although AH analytes were also quantifiable in smaller, less advanced tumors, AH samples from eyes with larger, more advanced tumors had higher analyte concentrations. The detection of prognostic biomarkers in AH may allow for early UM recognition and guide personalized treatment. Citation Format: Christina Chang, Sarah Pike, Mark Reid, Chen-Ching Peng, Benjamin Xu, Jesse Berry, Liya Xu. Nucleic acid quantification in uveal melanoma aqueous humor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3667.

Detection of doping substances in paired dried blood spots and urine samples collected during doping controls in Danish fitness centers.

The use of dried blood spot (DBS) in anti-doping can be advantageous in terms of collection, transportation, and storage compared with the traditional anti-doping testing matrices urine and venous blood. There could, nonetheless, be disadvantages such as shorter detection windows for some substances compared with urine, but real-life comparison of the detectability of prohibited substances in DBS and urine is lacking. Herein, we present a liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based screening method for simultaneous detection of 19 target analytes from the doping substance categories S1-S5 in a single spot. Ninety-eight urine and upper-arm DBS (Tasso-M20) sample pairs were collected from fitness centers customers notified for doping control by Anti Doping Denmark, and three sample pairs were collected from active steroid users undergoing clinical evaluation and treatment at a Danish hospital. The analytical findings were cross compared to evaluate the applicability of the developed DBS testing menu in terms of feasibility and analytical performance. To our knowledge, this is the first study to compare the detectability of prohibited substances in DBS and urine samples collected in a doping control setting. Twenty-seven of the urine samples and 23 DBS samples were positive, and we observed a very high concordance (95%) in the overall analytical results (i.e., positive or negative samples for both urine and DBS). Collectively, these results are very promising, and DBS seems suitable as a stand-alone matrix in doping control in fitness centers likely because of the high analyte concentration levels in these samples.

Slice through the water-Exploring the fundamental challenge of water suppression for benchtop NMR systems.

Benchtop NMR provides improved accessibility in terms of cost, space, and technical expertise. In turn, this encourages new users into the field of NMR spectroscopy. Unfortunately, many interesting samples in education and research, from beer to whole blood, contain significant amounts of water that require suppression in 1H NMR in order to recover sample information. However, due to the significant reduction in chemical shift dispersion in benchtop NMR systems, the sample signals are much closer to the water resonance compared to those in a corresponding high-field NMR spectrum. Therefore, simply translating solvent suppression experiments intended for high-field NMR instruments to benchtop NMR systems without careful consideration can be problematic. In this study, the effectiveness of several popular water suppression schemes was evaluated for benchtop NMR applications. Emphasis is placed on pulse sequences with no, or few, adjustable parameters making them easy to implement. These fall into two main categories: (1) those based on Pre-SAT including Pre-SAT, PURGE, NOESY-PR, and g-NOESY-PR and (2) those based on binomial inversion including JRS and W5-WATERGATE. Among these schemes, solvent suppression sequences based on Pre-SAT offer a general approach for easy solvent suppression for samples with higher analyte concentrations (sucrose standard and Redbull™). However, for human urine, binomial-like sequences were required. In summary, it is demonstrated that highly efficient water suppression approaches can be implemented on benchtop NMR systems in a simple manner, despite the limited spectral dispersion, further illustrating the potential for widespread implementation of these approaches in education and research.

Internationally standardized respiratory viral load testing with limited resources: A derivative-of-care calibration strategy for SARS-CoV-2.

SARS-CoV-2 has demonstrated that, in targeted circumstances, viral quantification within respiratory specimens can valuably inform patient management, as well as research. Nevertheless, the pandemic has illustrated concomitant challenges for obtaining high-quality (and broadly comparable) respiratory viral loads. This includes a critical need for standardization and calibration, even though the necessary resources may not always be available for emergent pathogens and non-bloodstream specimens. To these ends, we describe a novel strategy for implementing quantitative SARS-CoV-2 testing with International Unit-based calibration. Earlier in the course of the pandemic-when analytic resources were far more limited-select residual SARS-CoV-2 positive specimens from routine care in our diagnostic laboratory were pooled to formulate a clinically realistic secondary standard of high volume and analyte concentration, which was cross-calibrated to the primary SARS-CoV-2 standard of the World Health Organization. The resultant calibrators were integrated into the original CDC RT-qPCR assay for SARS-CoV-2, whose (now broadened) performance characteristics were defined to generate a test appropriate for both clinical and research use. This test allowed for the quantification of virus in respiratory specimens down to a validated lower limit of quantification of 103.4 IU/ml. By self-formulating calibrators from this derivative-of-care secondary standard, we successfully validated respiratory viral loads without the commercial availability (at that time) of quantitative assays or calibrators. As the SARS-CoV-2 pandemic continues to decline-and even beyond this pathogen-this strategy may be applicable for laboratories seeking to implement viral load testing for nontraditional specimen types despite limited resources.

On the Measurements of the Surface-Enhanced Raman Scattering Spectrum: Effective Enhancement Factor, Optical Configuration, Spectral Distortion, and Baseline Variation

In this paper, a comprehensive theoretical framework for understanding surface-enhanced Raman scattering (SERS) measurements in both solution and thin-film setups, focusing on electromagnetic enhancement principles, was presented. Two prevalent types of SERS substrates found in the literature were investigated: plasmonic colloidal particles, including spherical and spheroid nanoparticles, nanoparticle diameters, and thin-film-based SERS substrates, like ultra-thin substrates, bundled nanorods, plasmonic thin films, and porous thin films. The investigation explored the impact of analyte adsorption, orientation, and the polarization of the excitation laser on effective SERS enhancement factors. Notably, it considered the impact of analyte size on the SERS spectrum by examining scenarios where the analyte was significantly smaller or larger than the hot spot dimensions. The analysis also incorporated optical attenuations arising from the optical properties of the analyte and the SERS substrates. The findings provide possible explanations for many observations made in SERS measurements, such as variations in relative peak intensities during SERS assessments, reductions in SERS intensity at high analyte concentrations, and the occurrence of significant baseline fluctuations. This study offers valuable guidance for optimizing SERS substrate design, enhancing SERS measurements, and improving the quantification of SERS detection.

New Approaches to Stretched Film Sample Alignment and Data Collection for Vibrational Linear Dichroism.

Rapid measurements of vibrational linear dichroism (VLD) infrared spectra are shown to be possible by using stretched polymer films and an extension of existing instrumentation designed for vibrational circular dichroism spectroscopy. Earlier techniques can be extended using additional inexpensive polymer substrates to record good-quality VLD spectra of a significantly wider range of compounds with comparatively short sample-preparation times. The polymer substrates used, polyethylene and polytetrafluoroethylene, are commonly available and inexpensive, and samples are more easily prepared than that for many earlier stretched-film and crystal studies. Data are presented for neutral hydrophobic organic molecules on hydrophobic films including acridine, anthracene, fluorene, and recently synthesized S-(4-((4-cyanophenyl)ethynyl)phenyl)ethanethioate. We extend the approach to polar or ionic species, including 2,2'-bipyridine, 1,10-phenanthroline, and sodium dodecyl sulfate, by oxidizing polyethylene films to change their wetting properties. The combination of new instrumentation and modified sample preparation methods is useful in basic spectroscopy for untangling and assigning complicated infrared spectra. Nevertheless, it is not a panacea as surface-adsorbed molecules are often not monodispersed, and higher analyte concentrations can lead to aggregation and resonance phenomena that have previously been observed for infrared spectra on surfaces. These effects can be assessed by varying the sample concentration. The focus of this paper is experimental, and detailed analysis of most of the spectra lies outside its scope, including some well-studied compounds such as acridine and anthracene that allow comparisons with earlier research.

MOF-composite sensors to eliminate the QCM positive frequency shift

In a quartz crystal microbalance (QCM) Sauerbrey equation can usually be applied in the ideal scenario and when the QCM frequency shift goes to the negative direction. In some cases, however, the requirements to achieve the ideal condition are not fulfilled and thus leading to QCM positive frequency shift phenomenon. Herein we have also observed that a porphyrin-based metal organic frameworks (MOF), namely MOF-525(Zn), QCM sensor also exhibits the positive frequency shift phenomenon when exposed to xylene isomers, which could be caused by the loose attachment of the MOF nanoparticles on the QCM surface. Therefore, in the presence of the analytes, the bonding between the MOF and the QCM surface is disturbed and higher analyte concentration also leads to the increase of the sensitive layer stiffness, resulting in higher positive frequency shift. However, such a phenomenon can be completely eliminated by simply adding polymer to MOF nanoparticle sensors to strengthen the bonding of MOF nanoparticles to the QCM surface. Through this study we have shown that adjusting the amount of polymer is very crucial to completely reverse the positive frequency response. Otherwise, it appears a critical analyte concentration of gas analytes where the QCM frequency shift changes its direction from negative to positive response. Lastly, the same strategy has also been used to other MOF-based QCM sensor systems where their sensitive layer is built from other MOFs, namely MOF-525 and NU-902, and ethanol is used as the analyte to prove the generalizability of this simple approach.

Determination of Oseltamivir in Human Plasma by HPLC-MS/MS.

A procedure for the determination of oseltamivir in human plasma by high-performance liquid chromatography( tandem mass spectrometry (HPLC-MS/MS) was proposed and validated. Arapid and easy-to-use method of liquid(liquid extraction with ethyl acetate using venlafaxine as an internal standard was used during sample preparation. The addition of benzoic acid to aqueous acetonitrile solutions of the analyte was shown to prevent its oxidative degradation. The detection limit and limit of quantitation were 0.08 and 0.30 ng/mL, respectively; the calibration range, 0.3-200 ng/mL (R 2 = 0.9937); the total analysis time, 3.2 min. The within- and between-run accuracy ranged from 97 to 105%. The precision was <10%. The proposed procedure was characterized by selective determination of the analyte, the absence of significant matrix effects, the ability to dilute samples with high analyte concentrations, and satisfactory extraction recovery (≥89%). The analyte was stable when stored in plasma samples (4 h at room temperature, 31 d at (80°C, after three freeze(thaw cycles) and extracts under autosampler storage conditions (24 h at 15°C). The procedure was successfully used for oseltamivir quantitation in actual plasma samples from healthy volunteers obtained during a bioequivalence study of the new generic drug.

PEGylated Network Nanostructured by Gold Nanoparticles for Electrochemical Sensing of Aromatic Redox and Nonredox Analytes

We present the design, tailoring, and physicochemical properties of a multicomponent polyethylene glycol (PEG)-based polymer doped with spherical nanoparticles modified with a lipoic acid derivative of cyclodextrin and its application for the electrochemical sensing of a ferrocene derivative and ibuprofen at low concentrations. Each of the components of this hybrid material has specific properties that can be useful for the preparation of sensors characterized by high selectivity and a low detection limit. Electrochemical studies have previously shown that the cyclodextrin derivative with an aromatic triazole linker in the structure enhances binding of the analyte, ensuring its preconcentration on the electrode and, therefore, enabling improvement of the sensor detection limit. On the other hand, the introduction of evenly dispersed spherical gold nanoparticles in the polymer matrix increases the conductivity of the film and enhances the analytical signal. The third component─a biocompatible and easily wettable cross-linker, PEG─allows for uninterrupted diffusion within the polymer film. This significantly increases the analytical performance of the sensor, supports further improvement of the detection limit, and enables the determination of the analyte after saturation of cyclodextrin cavities (high concentration of analyte) based on its diffusion response. The designed hybrid material was used for the direct and indirect determination of aromatic analytes─ferrocene derivative and ibuprofen─with a very low detection limit.

Kinetic characterization of SPR-based biomarker assays enables quality control, calibration free measurements and robust optimization for clinical application

ABSTRACT: Biomarker measurements are essential for the early diagnosis of complex diseases. However, many current biomarker assays lack sensitivity and multiplexing capacity, work in a narrow detection range and importantly lack real time quality control opportunities, which hampers clinical translation. In this paper, we demonstrate a toolbox to kinetically characterize a biomarker measurement assay using Surface Plasmon Resonance imaging (SPRi) with ample opportunities for real time quality control by exploiting quantitative descriptions of the various biomolecular interactions. We show an accurate prediction of SPRi measurements at both low and high concentrations of various analytes with deviations <5% between actual measurements and predicted measurement. The biphasic binding sites model was accurate for fitting the experimental curves and enables optimal detection of heterophilic antibodies, cross-reactivity, spotting irregularities and/or other confounders. The toolbox can also be used to create a (simulated) calibration curve, enabling calibration-free measurements with good recovery, it allows for easy assay optimizations, and could help bridge the gap to bring new biomarker assays to the clinic.

Development of a Low-Cost Lateral Flow Assay for Rapid Detection of Vibrio Cholerae

Cholera is an acute, often fatal diarrheal disease that primarily affects areas with poor sanitation and unclean water. Cholera is caused by toxigenic strains of Vibrio cholerae (V. cholerae), which cause the body to rapidly dehydrate upon colonizing the intestine. The WHO estimates that 2.9 million people contract cholera each year, resulting in approximately 95000 deaths [1].Current methods for detection of V. cholerae are either inferential (detection of fecal coliforms or monitoring weather patterns) or diagnosed after an infection (stool tests) [2]. Even methods that have been developed for direct detection of V. cholerae fall short of detecting environmental concentrations of the bacterium (10-104 CFU/mL) [3]; a lateral flow assay (LFA) using gold nanoparticle-labelled antibodies for the simultaneous detection of O1 and O139 strains of V. cholerae achieved a limit of detection of 108 CFU/mL for O1 and 107 CFU/mL for O139 [4]. The current work aims to develop an LFA for the detection of V. cholerae at environmental conditions in drinking water under limited resource settings. By incorporating novel aptamers as antigen detecting agents, carbon nanoparticles as colorimetric indicators, and N-acetylglucosamine (GlcNAc)–a known chemoattractant for V. cholerae –in the sample pad, the limit of detection will be improved without the need for lengthy preprocessing steps or costly reagents.The antigen detection agent employed in the LFA is the aptamer V.ch27 which was identified by Mojarad and Gargaria [5]. This aptamer was shown to have a favourable secondary structure at 21°C and high binding affinity to the surface of V. cholerae bacteria. Aptamers are a synthetic alternative to conventionally used antibodies for this application. They exhibit specificities comparable to those of monoclonal antibodies, are cheaper to synthesize, and can be easily immobilized to surfaces via biotin-avidin complexing.Amorphous carbon nanoparticles (ACNPs) were chosen as the colorimetric indicator for the assay due to their strong black color and low cost [6]. For instance, carbon nanoparticles have shown a 100-fold decrease in the limit of detection for dengue fever antigen when compared to gold nanoparticles [7]. The ACNPs were synthesized by collecting the soot from burning candles followed by size selection by centrifugation, as previously described Zhang et al [8].The chemotaxis of V. cholerae to GlcNAc is well documented in literature [9]. The sample pad of the LFA contains a GlcNAc solution which diffuses into the water sample. V. cholerae swim up the GlcNAc concentration gradient and accumulate in high concentrations at the sample pad. This in turn results in a high concentration of analyte entering the assay, and consequently, a greater signal intensity. Previous studies have shown that chemotaxis can be exploited to collect nearly all of the bacteria in a 100 mL water sample [10,11].The fabricated LFA device will provide a rapid, accessible, and low-cost platform for V. cholerae detection in the water sources available to rural and low-income communities, without the need for technical training or well-established laboratory infrastructure. Successful implementation of the device will result in the overall reduction of global deaths due to cholera, as well as provide the framework for future preventative initiatives aimed towards cholera outbreak prediction and monitoring.

An analytical quality by design approach towards a simple and novel HPLC-UV method for quantification of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline

N-acetyl-seryl-aspartyl-lysyl proline (Ac-SDKP) is a tetrapeptide possessing anti-fibrotic, angiogenic, anti-inflammatory, anti-apoptotic, and immunomodulatory properties. Currently, the main method to quantify the peptide is liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), both of which are labour intensive and require expensive equipment and consumables. Furthermore, these techniques are generally utilised to detect very low or trace concentrations, such as in biological samples. The use of high concentrations of analyte might overload the extraction column or the separation column in LC-MS/MS or the ELISA plates, so the response could be a non-linear relationship at high analyte concentrations. Thus, they are not ideal for formulation development where detection of dose-equivalent concentrations is typically required. Therefore, a cost-effective, simple, and accurate quantification method for the peptide at a higher concentration needs to be developed. In this study, a simple and novel HPLC-UV method is proposed and validated using an Analytical Quality by Design (AQbD) approach. The method is first screened and optimised using chromatographic responses including capacity factor, resolution, tailing factor, and theoretical plate counts, fulfilling the International Council for Harmonisation (ICH) Q2 (R1) guidelines. The resultant optimised chromatography conditions utilised 10 mM phosphate buffer at pH 2.5 and acetonitrile as mobile phases, starting at 3% (v/v) acetonitrile and 97% (v/v) buffer and increasing to 9.7% (v/v) acetonitrile and 90.3% (v/v) buffer over 15 min at a flow rate of 1 mL/min at the column temperature of 25 °C. The injection volume is set at 10 μL and the VWD detector wavelength is 220 nm. The method established is suitable for detecting the peptide at a relatively high concentration, with a quantifiable range from 7.8 μg/mL to 2.0 mg/mL. In addition, the use of a relatively simple HPLC-UV approach could significantly reduce costs and allow easier access to quantify the peptide concentration. A limitation of this method is lower sensitivity compared with using LC-MS/MS and ELISA methods but running costs are lower and the methodology is simpler. The method is capable to quantify the peptide in various tested matrix solutions, with successful quantitation of the peptide in samples obtained from in vitro drug release study in PBS and from a chitosan-TPP nanogels formulation. Therefore, the method developed here offers a complementary approach to the existing quantification methods, quantifying this peptide at increased concentrations in simple to intermediately complex matrix solutions, such as HBSS, DMEM and FluoroBrite cell culture media.

Abstract 3919: MosaicNeedles: A tool for large-scale proteomics combining antibodies and aptamers

Abstract Proteomics have the potential to transform biomarker discovery, diagnostics, and personalized medicine as it provides functional and actionable information linked to disease phenotypes. However, mass spectrometry (MS) – based proteomics has not been possible at large scale due to limited dynamic range, complex workflow and high experimental cost per sample. Antibody-based methods, although widely used in targeted proteomics for their better sensitivity, have been limited to the profiling of a few hundred selected proteins, partly due to the limited availability of antibody pairs. NanoMosaic has demonstrated antibody-aptamer sandwich assays on the MosaicNeedle࣪ platform. This expands the content size from a few hundred to a few thousand, achievable in one reaction with high sensitivity and specificity and low cross-reactivity. MosaicNeedles࣪ are densely integrated nanoneedle sensors fabricated on a planar substrate. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can also be grouped into 96, 384 or 1536 formats. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture antibodies or aptamers. At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. We have demonstrated that the aptamer-antibody assay on the MosaicNeedle࣪ platform achieves better sensitivity than the well-developed antibody-pair assay. It also achieves larger dynamic range and alleviates the antibody cross-reactivity by refining aptamers during the SELEX process. The scalability in manufacturing nanoneedles combined with the low sample requirement and no fluorescent labeling all help to drive down the cost-per-protein to enable large-scale, unbiased proteomics. We envision researchers will use MosaicNeedle࣪ platform for both the upstream target discovery phase and then transfer seamlessly to the downstream validation phase by building customized and targeted panels for proteins of high interest that emerge from the discovery phase. Citation Format: Qimin Quan, Alaina Kaiser, Joshua Ritchey, Joe Wilkinson, John Geanacopoulos, John Boyce. MosaicNeedles: A tool for large-scale proteomics combining antibodies and aptamers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3919.

Ultrastable Lactate Biosensor Linearly Responding in Whole Sweat for Noninvasive Monitoring of Hypoxia.

We report on the lactate biosensor with linear calibration range from 0.5 to 100 mM, which encircles possible levels of this metabolite concentration in both human sweat and blood. The linear calibration range at high analyte concentrations, which exceeds the Michaelis constant of lactate oxidase by several orders of magnitude, is provided by an additional perfluorosulfonated ionomer diffusion membrane. In contrast to the known lactate biosensors, which retain their response within less than a couple of hours, the reported system displays 100% response for dozens of hours even upon high analyte concentrations. The biosensors with an additional diffusion-limiting membrane have been validated for lactate detection both in human blood serum and in undiluted human sweat shortly after its secretion. Both linear response in the entire range of blood and sweat lactate concentrations and ultrahigh operational stability would provide the use of the elaborated biosensor in wearable devices for the monitoring of hypoxia.

Proteogenomic detection of circulating biomarkers for clinical oncology.

e15010 Background: Circulating biomarkers have the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteogenomics in circulating biomarkers may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. Proteomics bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in a genomic approach. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: MosaicNeedles are densely integrated nanoneedle sensors fabricated on a planar substrate that integrates proteogenomic analysis in one platform. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can be configured into 96, 384 or 1536 well format. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. Results: At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: A full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction with high sensitivity and large dynamic range. It simplifies the workflow and allows users to integrate proteomic and genomic information to discover new circulating biomarkers.

Mitigating hook effect in one-step quantitative sandwich lateral flow assay by timed conjugate release.

Sandwich lateral flow assay (LFA) is one of the most successfully commercialized paper-based biosensors, which offers a rapid, low-cost, one-step assay. Despite its advantages, conventional sandwich LFA is fundamentally limited by the high-dose "hook" effect-a phenomenon that occurs at very high analyte concentrations and results in false-negative results. In this paper, we present a novel strategy of automatic timed detection antibody release to mitigate the hook effect in sandwich LFA without additional manual steps. We introduced an intermediate pad treated with saturated sucrose solution to regulate the flow between the nitrocellulose membrane and the conjugate pad in order to delay the reaction between detection antibodies and analytes. Using C-reactive protein (CRP) as a representative analyte, we demonstrated that our strategy exhibited a range of detection 10 times wider than that of our conventional LFA, without sacrificing the limit of detection. Comparing to other published strategies, our work could offer a one-step, cost-effective approach that is closely unified with the benefits of the LFA.

Analytical validation of 39 clinical chemistry tests and 17 immunoassays on the Alinity analytical system

The aim of this study was to perform the analytical validation of Alinity c and i analyzers (Abbott Laboratories, Chicago, IL, USA) for 39 clinical chemistry tests and 17 immunoassays. Precision was evaluated at least at two concentration levels for 5 days in quintuplicate, following CLSI EP15-A3. Method comparison included parallel analysis of leftover routine samples on Alinity analyzers and the previously used Cobas c501 and e601 (Roche Diagnostics, Mannheim, Germany). Linearity was tested by preparing sequential sample dilutions with high analyte concentration, following the CLSI EP6 document. For clinical chemistry tests, within-run coefficients of variation (CV) were up to 6.0% (beta-2-microglobulin), while between-run CVs up to 5.4% (immunoglobulin M). Among immunoassays, the highest within-run CV was obtained for vitamin B12 (6.9%), while between-run for CA 19-9 (4.3%). Complete agreement with Roche analyzers was observed for 16 (41%) clinical chemistry assays and 6 (35%) immunoassays. Half of all evaluated assays did not meet the desirable biological variation criteria for bias, being especially exceeded for alpha1-antitrypsin, apolipoprotein A1, ceruloplasmin, complement C3 and C4, hemoglobin A1c, lipoprotein (a) and myoglobin, as well as some tumor markers (CA 125, CEA, fPSA, AFP, and ferritin), hormones (cortisol, DHEA-S, insulin) and vitamins (25-OHD). Linearity in the tested ranges was confirmed. Overall, this study revealed that precision criteria derived from manufacturer's claims were not satisfied for all assays while comparison study for some assays yielded differences that imply the need for additional assay evaluation prior to introduction into routine practice.

Suppressing Nonspecific Binding in Biolayer Interferometry Experiments for Weak Ligand-Analyte Interactions.

Quantitative analysis of protein–protein interactions (PPIs) using biolayer interferometry (BLI) requires effective suppression of nonspecific binding (NSB) between analytes and biosensors. In particular, the study of weak interactions (i.e., KD > 1 μM) requires high concentrations of analytes, which substantially increases NSB. However, there are only a few so-called NSB blockers compatible with biomolecules, which limits the use of BLI in the accurate analysis of weak interactions. The present study aims to identify a new NSB blocker for the quantitative analysis of weak PPIs using BLI. We find that saccharides, especially sucrose, are potent NSB blockers and demonstrate their compatibility with other blocking additives. We also demonstrate the effects of the new NSB blocker by characterizing the binding between nonstructural protein 1 of the influenza A virus and human phosphoinositide 3-kinase. We anticipate that the new NSB-blocking admixture will find broad applications in studying weak interactions using BLI.