Broad Dynamic Range Research Articles

Optimization and miniaturization of SE-ECL for potential-resolved, multi-color, multi-analyte detection

Electrochemiluminescence (ECL) is a bioanalytical technique with numerous advantages, including the potential for high temporal and spatial resolution, a high signal-to-noise ratio, a broad dynamic range, and rapid measurement capabilities. To reduce the complexity of a multi-electrode approach, we use a single-electrode electrochemiluminescence (SE-ECL) configuration to achieve the simultaneous emission and detection of multiple colors for applications that require multiplexed detection of several analytes. This method exploits intrinsic differences in the electric potential applied along single electrodes built into electrochemical cells, enabling the achievement of distinct colors through selective excitation of ECL luminophores. We present results on the optimization of SE-ECL intensity for different channel lengths and widths, with sum intensities being 5 times larger for 6 cm vs. 2 cm channels and linearly increasing with the width of the channels. Furthermore, we demonstrated for the first time that applying Alternating Current (AC) voltage within the single electrode setup for driving the ECL reactions has a dramatic effect on the emitted light intensity, with square waveforms resulting in higher intensities vs sine waveforms. Additionally, multiplexed multicolor SE-ECL on a 6.5 mm × 3.6 mm CMOS semiconductor image sensor was demonstrated for the first time, with the ability to simultaneously distinguish four different colors, leading to the ability to measure multiple analytes.

Development and validation of ELISA method for quantification of Q-1802 in serum and its application to pharmacokinetic study in ICR Mouse

Q-1802 is a humanized bispecific antibody targeting programmed death-ligand 1 (PD-L1) and Claudin 18.2 (CLDN18.2). It can bind to CLDN18.2 and mediate antibody-dependent cell-mediated cytotoxicity against tumor cells. The Fc segment of the antibody recognizing PD-L1 blocks PD-1 signaling and activates innate immunity and adaptive immunity. In this study, we report the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of Q-1802 in ICR mouse serum. The assay is sensitive, with a lower limit of quantification of 50 ng/mL, has a broad dynamic range of 50–3200 ng/mL, and exhibits excellent precision and accuracy. These assays were successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring Q-1802 in ICR mouse serum. The development and validation steps of assays met the required criteria for validation, which suggested that these can be applied to quantify Q-1802, as well as in PK studies.

Development of a novel fluorescent protein-based probe for efficient detection of Pb2+ in serum inspired by the metalloregulatory protein PbrR691

BackgroundThe accurate and rapid detection of blood lead concentration is of paramount importance for assessing human lead exposure levels. Fluorescent protein-based probes, known for their high detection capabilities and low toxicity, are extensively used in analytical sciences. However, there is currently a shortage of such probes designed for ultrasensitive detection of Pb2+, and no reported probes exist for the quantitative detection of Pb2+ in blood samples. This study aims to fill this critical void by developing and evaluating a novel fluorescent protein-based probe that promises accurate and rapid lead quantification in blood. ResultsA simple and small-molecule fluorescent protein-based probe was successfully constructed herein using a peptide PbrBD designed for Pb2+ recognition coupled to a single fluorescent protein, sfGFP. The probe retains a three-coordinate configuration to identify Pb2+ and has a high affinity for it with a Kd’ of 1.48 ± 0.05 × 10−17 M. It effectively transfers the conformational changes of the peptide to the chromophore upon Pb2+ binding, leading to fast fluorescence quenching and a sensitive response to Pb2+. The probe offers a broad dynamic response range of approximately 37-fold and a linear detection range from 0.25 nM to 3500 nM. More importantly, the probe can resist interference of metal ions in living organisms, enabling quantitative analysis of Pb2+ in the picomolar to millimolar range in serum samples with a recovery percentage of 96.64%–108.74 %. SignificanceThis innovative probe, the first to employ a single fluorescent protein-based probe for ultrasensitive and precise analysis of Pb2+ in animal and human serum, heralds a significant advancement in environmental monitoring and public health surveillance. Furthermore, as a genetically encoded fluorescent probe, this probe also holds potential for the in vivo localization and concentration monitoring of Pb2+.

Microarray Platforms Based on 3D Printing.

This paper introduces an innovative method for the fabrication and infusion of microwell arrays based on digital light processing (DLP) 3D printing. A low-cost DLP 3D printer is employed to fabricate microstructures rapidly with a broad dynamic range while maintaining high precision and fidelity. We constructed microwell arrays with varying diameters, from 200 to 2000 μm and multiple aspect ratios, in addition to microchannels with widths ranging from 45 to 1000 μm, proving the potential and flexibility of this fabrication method. The superimposition of parallel microchannels onto the microwell array, facilitated by positive or negative pressure, enabled the transfer of liquid to the microwells. Upon removal of the microchannel chip, a dispensed microdroplet array was obtained. This array can be modulated by adjusting the volume of the microwells and the inflow fluid. The filled microwell array allows chip-to-chip dispensing to the microreactor array through binding and centrifugation, facilitating multistep and multireagent assays. The 3D printing approach also enables the fabrication of intricate cavity designs, such as micropyramid arrays, which can be integrated with parallel microchannels to generate spheroid flowcells. This device demonstrated the ability to generate spheroids and manipulate their environment. We have successfully utilized precise modulation of spheroids size and performed parallel drug dose-response assays to evaluate its effectiveness. Furthermore, we managed to execute dynamic drug combinations based on a compact spheroids array, utilizing two orthogonal parallel microchannels. Our findings suggest that both the combination and temporal sequence of drug administration have a significant impact on therapeutic outcomes.

Optimization of periodic treatment strategies for bacterial biofilms using an agent-based in silico approach.

Biofilms are responsible for most chronic infections and are highly resistant to antibiotic treatments. Previous studies have demonstrated that periodic dosing of antibiotics can help sensitize persistent subpopulations and reduce the overall dosage required for treatment. Because the dynamics and mechanisms of biofilm growth and the formation of persister cells are diverse and are affected by environmental conditions, it remains a challenge to design optimal periodic dosing regimens. Here, we develop a computational agent-based model to streamline this process and determine key parameters for effective treatment. We used our model to test a broad range of persistence switching dynamics and found that if periodic antibiotic dosing was tuned to biofilm dynamics, the dose required for effective treatment could be reduced by nearly 77%. The biofilm architecture and its response to antibiotics were found to depend on the dynamics of persister cells. Despite some differences in the response of biofilm governed by different persister switching rates, we found that a general optimized periodic treatment was still effective in significantly reducing the required antibiotic dose. As persistence becomes better quantified and understood, our model has the potential to act as a foundation for more effective strategies to target bacterial infections.

Abstract 936: Targeted proteomics and next-generation sequencing (NGS) for biomarker discovery in metastatic colorectal cancer (mCRC)

Abstract Background: Multi-omics analysis is widely used in cancer diagnosis, prognosis and prediction of treatment response. Here we introduce the use of targeted proteomics coupled with NGS as tools to assess oncogenic alterations along with quantitative protein expression in patients with mCRC. Our proteomics panel included proteins that are antibody drug conjugates (ADCs) targets (ERBB 1-4, TROP2, CEACAM5 etc.) as well as proteins involved in sensitivity to ADC payload (SLFN11, drug efflux pump, TOP1 etc.). Methods: The HORIZON III trial (NCT00384176) evaluated FOLFOX + bevacizumab or cediranib as first-line treatment in patients with mCRC (Schmoll J Clin Oncol 2012). In the current analysis, a subset of samples from the HORIZON III trial were assessed for molecular alterations using NGS and proteomics. Pre-existing HER2 IHC scores were used as previously referenced (Cecchi J Clin Oncol 2023). Samples were processed (n=135) with an automated extraction and library preparation method, then sequenced with both a Whole Exome and targeted-custom gene panel. NGS analysis had a success rate of 116/135 samples (87.3%). Absolute quantitation of protein targets was performed using parallel reaction monitoring (PRM) mass spectrometry after collecting tumor epithelial cells by laser microdissection and punch biopsies (n=60) followed by trypsin digestion. Results: The NGS analysis showed that patients had mutated RAS (50%), BRAF (10%), PIK3CA (16%), and TMB-H (18%) and MSI-H (5%) status. A distinguished pattern of mutual exclusivity among KRAS, NRAS, and BRAF mutations is evident. The HER2-positive patients were wildtype RAS, BRAF, and PIK3CA, MSS and TMB-L. Samples that were IHC HER2-positive matched those with NGS ERBB2 amplification. Using PRM, nearly all the evaluable samples had detectable HER2. HER2 IHC 3+ sample showed the highest protein expression at 30,000 amol/ug (equivalent to 1M receptors/cell). Within the HER2-negative patients, HER2 protein expression showed broad dynamic range. CEACAM5 and HER2 are the only ADC targets that reached the level of 1M receptors/cell. Highest TROP2 protein expression is 5 times lower than the highest HER2 value. Conclusions: Our preliminary findings for this cohort indicate the benefit of the use of multi-modal methodologies to inform better strategies for patient stratification and precision medicine, though further assessment of proteogenomic characteristics is needed. This data may inform future clinical development and guide patient eligibility to clinical trials and possible treatment sequencing for ADCs. Citation Format: William J. Kelly, Barrett Nuttall, Marlon Rebelatto, Marcus Schick, Armin Meier, Steve Sweet, Beom-Jun Kim, David Chain, Yeoun Jin Kim, Zhongwu Lai, Ying Wang, Todd H. Creasy, Ramya Gurrapu, Sakshi Gulati, Alice Barkell, Brian Dougherty, Danielle Carroll, Fabiola Cecchi. Targeted proteomics and next-generation sequencing (NGS) for biomarker discovery in metastatic colorectal cancer (mCRC) [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 936.

Abstract 2908: EDB+FN is an attractive therapeutic target in oncology: Insights from protein expression analysis of solid tumors

Abstract Stroma plays a major role in the initiation, growth, survival, and drug-resistance of solid tumors, yet few therapeutics specifically target tumor-associated stroma. Extra-domain-B fibronectin (EDB+FN) is an oncofetal splice variant of fibronectin upregulated in solid tumor stroma, which is associated with tumor growth, angiogenesis, and metastases. Here we developed an immunohistochemistry (IHC) assay to assess EDB+FN protein expression in the stroma of tumor and normal tissues to characterize the potential of EDB+FN as a therapeutic target for solid tumors with high unmet need. We validated a primary detection antibody and titrated assay conditions to achieve specificity and a broad dynamic range of EDB+FN expression levels across indications, including thyroid cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, soft tissue sarcoma, pancreatic cancer, non-small cell lung cancer, ovarian cancer, and renal cancer. Considering the stroma-specific expression pattern of EDB+FN, we developed a scoring algorithm weighting both the intensity and distribution of EDB+FN expression specifically in the stroma compartment and separately in tumor cells. Using this assay and scoring approach, we screened more than 200 tumor samples from 10 solid tumor indications and found EDB+FN to be highly differentially expressed in tumor-associated stroma with little to no expression on tumor cells and trace to undetectable levels in healthy tissue. EDB+FN was expressed across the indications evaluated with a wide range of expression both within and between indications. Stromal EDB+FN expression was not correlated with stromal density, which also varied widely within and across indications. In parallel, we developed an algorithm using digital pathology to approximate pathologist scoring of stromal density and EDB+FN expression, allowing streamlined screening of preclinical samples for in vivo studies and indication selection. The disease-specific expression pattern of EDB+FN and high incidence of expression across indications make EDB+FN an ideal target for high-unmet-need solid tumors. The specificity and dynamic range of our IHC assay for detection of EDB+FN protein expression position it as a robust potential biomarker assay for EDB+FN-targeting therapeutics. Citation Format: Sara Lewandowski, Liyang Diao, Alyssa Quigley, Marsha Crochiere, Jan Pinkas. EDB+FN is an attractive therapeutic target in oncology: Insights from protein expression analysis of solid tumors [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 2908.

Abstract 2535: Development of Claudin 18.2 IHC 14G11 pharmDx as a clinical trial assay for the clinical development of anti-claudin 18.2 monoclonal antibody osemitamab (TST001) in gastric and gastroesophageal junction adenocarcinoma

Abstract Clone 14G11 is a mouse anti-Claudin 18.2 monoclonal antibody developed by Transcenta. The antibody was generated against a linear epitope located on the extracellular domain of loop1 and has a binding site that overlaps with the binding site of therapeutic antibody Osemitamab. It was selected to further develop for the detection of Claudin 18.2 expression in human cancer. Agilent Technologies, Inc. is developing Claudin 18.2 IHC 14G11 pharmDx as an immunohistochemical (IHC) assay for the detection of Claudin 18.2 protein in gastric and gastroesophageal junction (GEJ) adenocarcinoma in support of clinical studies conducted by Transcenta Therapeutics. Analytical verification for Claudin 18.2 IHC 14G11 pharmDx included sensitivity, specificity, and precision assessment. Testing was performed on human formalin-fixed, paraffin-embedded (FFPE) gastric and GEJ tissues using Autostainer Link 48 with EnVision FLEX visualization system. Biomarker expression was evaluated using Tumor Proportion Score (TPS). TPS is defined as the percentage of viable invasive tumor cells that exhibit convincing partial or complete membrane staining at any intensity. Sensitivity testing of 102 commercially procured FFPE gastric/GEJ specimens demonstrated a broad dynamic range of Claudin 18.2 positivity that covered TPS from 0% to 100%. Specificity tests confirmed specific targeting of the Claudin 18.2 protein in a variety of normal and abnormal tissues. For laboratory precision testing, which evaluated the assay variability in relation to run, instrument, operator, and reagent lot, ≥ 95% of TPS values were within expected variability limits for each parameter. For observer precision testing, two-sided 95% confidence intervals were ≥ 85% for negative, positive, and overall agreements when assessing two clinical cutoffs. These results demonstrated the Claudin 18.2 IHC 14G11 pharmDx assay can provide reliable and reproducible results in day-to-day testing. Analytical verification testing indicates Claudin 18.2 IHC 14G11 pharmDx is a sensitive, specific, and precise assay for detecting Claudin 18.2 in gastric and GEJ adenocarcinoma. Claudin 18.2 IHC 14G11 pharmDx for Investigational Use Only/for Performance Evaluation Only will be used for patient selection in the upcoming phase III trial of gastric/GEJ adenocarcinoma where applicable ethics committee and regulatory approvals have been granted. Citation Format: Hayley Alderson, Katie Lefley, Lesly Fregoso, Achintya Bharadwaj, Dan Castellanos-McKenney, Siena Tabuena-Frolli, Donna Kell, Xinlai Yao, Xueming Qian, Li Xu, Caroline Germa, Emin Oroudjev. Development of Claudin 18.2 IHC 14G11 pharmDx as a clinical trial assay for the clinical development of anti-claudin 18.2 monoclonal antibody osemitamab (TST001) in gastric and gastroesophageal junction adenocarcinoma [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 2535.

Abstract 3249: Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum

Abstract Adeno-associated viruses (AAV) are prevalent gene therapy vectors, delivering therapeutic transgenes to treat various diseases including cancer and rare monogenic diseases. Individuals with pre-existing immunity to AAVs are less likely to benefit from AAV-based therapies due to both, a reduction in cellular AAV uptake and activation of a cytotoxic T-cell response to transduced cells, decreasing efficacy or changing the safety profile. Measurement of serum neutralizing antibodies (NAbs) against AAV is essential to determine treatment eligibility and to identify clinical trial candidates. Here we introduce a cell-based assay using NanoLuc® luciferase to detect NAbs in serum samples. The superior brightness of NanoLuc® luciferase in our AAV reporter system results in the need of low concentrations of AAV and a short 24h assay time. The minimal AAV concentration in our assay aids in detecting low NAb titers in serum samples, reducing false negatives and enhancing decision-making accuracy. Testing 60 human serum samples against a range of AAV serotypes, we found that a large fraction of the population had pre-existing NAbs to at least one serotype, and of the pre-exposed individuals a majority displayed NAbs against multiple AAV serotypes. Interestingly, fewer samples were seropositive for AAV-DJ than for either parental serotype (AAV2, AAV8 or AAV9), highlighting the assays utility in the development of engineered capsids. Furthermore, the broad dynamic range of our assay facilitates serum sample categorization into four NAb tiers: negative, low, medium, and high using a single serum dilution. Neutralizing titers (ND50) which was determined by serial dilution of the serum samples strongly agreed with the assigned NAb tiers. Our study with 40 naïve mice further validated the assay's robustness, showing a clear correlation between NAb levels and injected AAV viral loads. Taken together, this rapid, highly sensitive, and reliable assay enabled by NanoLuc® AAV reporter technology precisely measures NAbs against AAVs in both human and animal serum. Further validation of the assay is required for future use in a clinical setting. Citation Format: Morten Seirup, Kirsten Wingate, Jeff Nelson, Kara Machleidt, Vanessa Ott, Trish Hoang, Marjeta Urh. Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum [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 3249.

A prototype scintillator real-time beam monitor for ultra-high dose rate radiotherapy.

FLASH Radiotherapy (RT) is an emergent cancer RT modality where an entire therapeutic dose is delivered at more than 1000 times higher dose rate than conventional RT. For clinical trials to be conducted safely, a precise and fast beam monitor that can generate out-of-tolerance beam interrupts is required. This paper describes the overall concept and provides results from a prototype ultra-fast, scintillator-based beam monitor for both proton and electron beam FLASH applications. A FLASH Beam Scintillator Monitor (FBSM) is being developed that employs a novel proprietary scintillator material. The FBSM has capabilities that conventional RT detector technologies are unable to simultaneously provide: (1) large area coverage; (2) a low mass profile; (3) a linear response over a broad dynamic range; (4) radiation hardness; (5) real-time analysis to provide an IEC-compliant fast beam-interrupt signal based on true two-dimensional beam imaging, radiation dosimetry and excellent spatial resolution. The FBSM uses a proprietary low mass, less than 0.5mm water equivalent, non-hygroscopic, radiation tolerant scintillator material (designated HM: hybrid material) that is viewed by high frame rate CMOS cameras. Folded optics using mirrors enable a thin monitor profile of ∼10cm. A field programmable gate array (FPGA) data acquisition system generates real-time analysis on a time scale appropriate to the FLASH RT beam modality: 100-1000Hz for pulsed electrons and 10-20kHz for quasi-continuous scanning proton pencil beams. An ion beam monitor served as the initial development platform for this work and was tested in low energy heavy-ion beams (86Kr+26 and protons). A prototype FBSM was fabricated and then tested in various radiation beams that included FLASH level dose per pulse electron beams, and a hospital RT clinic with electron beams. Results presented in this report include image quality, response linearity, radiation hardness, spatial resolution, and real-time data processing. The HM scintillator was found to be highly radiation damage resistant. It exhibited a small 0.025%/kGy signal decrease from a 216 kGy cumulative dose resulting from continuous exposure for 15 min at a FLASH compatible dose rate of 237Gy/s. Measurements of the signal amplitude versus beam fluence demonstrate linear response of the FBSM at FLASH compatible dose rates of >40Gy/s. Comparison with commercial Gafchromic film indicates that the FBSM produces a high resolution 2D beam image and can reproduce a nearly identical beam profile, including primary beam tails. The spatial resolution was measured at 35-40µm. Tests of the firmware beta version show successful operation at 20000Hz frame rate or 50 µs/frame, where the real-time analysis of the beam parameters is achieved in less than 1 µs. The FBSM is designed to provide real-time beam profile monitoring over a large active area without significantly degrading the beam quality. A prototype device has been staged in particle beams at currents of single particles up to FLASH level dose rates, using both continuous ion beams and pulsed electron beams. Using a novel scintillator, beam profiling has been demonstrated for currents extending from single particles to 10 nA currents. Radiation damage is minimal and even under FLASH conditions would require ≥50 kGy of accumulated exposure in a single spot to result in a 1% decrease in signal output. Beam imaging is comparable to radiochromic films, and provides immediate images without hours of processing. Real-time data processing, taking less than 50 µs (combined data transfer and analysis times), has been implemented in firmware for 20kHz frame rates for continuous proton beams.

Engineering and application of LacI mutants with stringent expressions.

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).

Dual Interface Modification Using Potassium Aspartic Acid to Realize Low Dark Current, High-Speed Nonfullerene Photodetectors.

Organic photodetectors (OPDs) have attracted tremendous interest due to their potential applications in wearable electronics. However, due to the nonideal contacts between the electrodes and organic semiconductors, OPDs still suffer from high dark current and slow frequency response. Herein, by inserting potassium aspartic acid (PAA) interlayers between ITO/metal oxides and the metal oxides/active layer, the shunts and hole injections are blocked and the energy levels of the electrodes are aligned. As a result, our dual-interface modified OPDs (ITO/PAA/ZnO/PAA/PTB7-Th:ITIC/MoO3/Ag) exhibit suppressed dark current 550 times lower than the reference device, corresponding to specific detectivity of 2.1 × 1012 Jones, broad linear dynamic range of 113 dB, and quick response time to the nanosecond level. PAA interlayers have also been demonstrated to improve the storage stability of OPDs, leading to 10 times slower degradation for the on/off ratio when compared with the reference and conventional polyethylenimine-modified OPDs.

Fabrication of an electrochemical impedance sensor for ketamine hydrochloride and its application in the detection of doping substances in sports

This work reports an ultrasensitive impedimetric immunosensor for ketamine detection using self-assembled monolayers of 3-mercaptopropionic acid on gold electrodes. Monoclonal ketamine antibodies were covalently tethered via EDC/NHS crosslinking chemistry. Each fabrication step was validated by electrochemical and microscopic characterization. The optimized immunoelectrode demonstrated a broad dynamic range from 1 to 1000 ng/mL ketamine and record low detection limit of 0.5 ng/mL attributed to excellent antibody affinity. Analytical recovery of spiked serum and urine samples ranged 92–103% proving reliability despite biosample complexity. The simple, rapid and portable sensor surpasses existing chromatography and electrochemical techniques in terms of detection sensitivity as well as cost, instrumentation and training needs. The capabilities are attributed to rational antibody immobilization strategies enabling efficient binding events. The attractive detection limits coupled with label-free operation and microscale construct provide optimism regarding translation to viable clinical and on-site testing devices for toxicology, anti-doping and therapeutic applications.

ChipFilter: Microfluidic-Based Comprehensive Sample Preparation Methodology for Microbial Consortia.

The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.

Biosensor-assisted CRISPRi high-throughput screening to identify genetic targets in Zymomonas mobilis for high d-lactate production

Lactate is an important monomer for the synthesis of poly-lactate (PLA), which is a substitute for the petrochemical plastics. To achieve the goal of high lactate titer, rate, and yield for commercial production, efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance. In this study, an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence, which could be the mutant strains with increased d-lactate production. Then, LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference (CRISPRi) library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome. Specifically, two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting (FACS), respectively. Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production, further knockout of ZMO1323 and ZMO1530 resulted in a 15% and 21% increase of d-lactate production, respectively. This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z. mobilis, but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

Electrostatically formed nanowire (EFN) transistor—An ultrasensitive VOC and gas sensor

The perpetual need for high-performance volatile organic compound (VOC) sensors remains prevalent across diverse sectors including environmental health monitoring, industrial operations, and medical diagnostics. Within this context, the electrostatically formed nanowire (EFN) sensor, a silicon-on-insulator-based multiple-gate field-effect transistor, is an ultrasensitive and selective VOC and gas sensing platform. Unlike conventional silicon nanowires (also known for their superior sensitivity to chemical species), in EFN, the nanowire is defined electrostatically post-fabrication through appropriate biasing of the surrounding gates. The fabrication of the EFN leverages established CMOS compatible silicon processing technologies, facilitating the production of inexpensive, scalable, and robust sensors. By precisely controlling gate biases, a conductive channel with a tunable diameter is formed, allowing for the formation of nanowire with diameter below 20 nm. The adjustable size and shape of the nanowire offer tunable sensing parameters, including sensitivity, limit of detection, and dynamic range. The multiple parameters also yield a unique fingerprint for each VOC, thus enabling selective detection of VOCs. By simply altering the biasing configuration, a single EFN sensor can achieve high sensitivity and a broad dynamic range, which is limited in the case of physically defined silicon NW sensors. This review provides a comprehensive overview encompassing the EFN sensor's design, fabrication considerations, process flow, electrical characterization methods, sensing performances to VOCs, and gases at room temperature. Moreover, the scope of advanced sensor designs with array of EFN sensors and integrated heaters is also discussed. Finally, some future perspectives of this technology are presented.

Packaged WGM MBR sensor for high-performance temperature measurement using CNN-based multimode barcode images.

The whispering gallery mode (WGM) optical microresonator sensors are emerging as a promising platform for precise temperature measurements, driven by their excellent sensitivity, resolution and integration. Nevertheless, challenges endure regarding stability, single resonant mode tracking, and real-time monitoring. Here, we demonstrate a temperature measurement approach based on convolutional neural network (CNN), leveraging the recognition of multimode barcode images acquired from a WGM microbottle resonator (MBR) sensor with robust packaged microresonator-taper coupling structure (packaged-MTCS). Our work ensures not only a high sensitivity of -14.28 pm/℃ and remarkable resolution of 3.5 × 10-4 ℃ across a broad dynamic range of 96 ℃ but also fulfills the demands for real-time temperature measurement with an average detection accuracy of 96.85% and a speed of 0.68s per image. These results highlight the potential of high-performance WGM MBR sensors in various fields and lay the groundwork for stable soliton microcomb excitation through thermal tuning.

Strain-enhanced dynamic ranges in two-dimensional MoS2 and MoTe2 nanomechanical resonators

Two-dimensional (2D) materials are promising for atomic-scale, ultralow-power, and highly tunable resonant nanoelectromechanical systems (NEMS) in sensing, communications, and computing. Toward these applications, a broad and controllable linear dynamic range (DR) is desirable for increasing the signal-to-noise ratio (SNR) and reliability. Here, we develop a comprehensive strain-enhanced DR model for 2D NEMS resonators, which is experimentally verified through the tuning of DRs in 2D molybdenum disulfide (MoS2) and molybdenum ditelluride (MoTe2) NEMS resonators using gate-induced strain. We find that the resonance frequency, quality factor, and nonlinear coefficient are all tuned by the gate voltage, which enhance the DR together. Through the guidance of the DR tuning model, we demonstrate DR enhancement by up to 26.9 dB (from 69.5 to 96.4 dB) in a 2D MoS2 NEMS resonator by properly tuning the gate voltage, leading to a theoretical mass resolution of 26 yg (1 yg = 10−24 g). To accurately extract the DR, we further differentiate the quality factors for thermomechanical resonances and for resonances at the largest linear amplitude. This gate-enhanced DR model is also verified using a MoTe2 resonator, with DR enhancement of 7 dB (91.2 to 98.2 dB). The results provide a promising pathway for accurately predicting and optimizing the DRs in NEMS resonators, toward enhanced sensitivity and SNR in mass sensing, radio frequency signal processing, memory, and computing applications.

Characterization of Novel Human β-glucocerebrosidase Antibodies for Parkinson's Disease Research.

Mutations in GBA1, which encodes the lysosome enzyme β-glucocerebrosidase (also referred to as acid β-glucosidase or GCase), are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme developed to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

Performance-enhanced intrinsic polarization-sensitive organic photodetectors by molecular interaction modulation

Polarization-sensitive organic photodetectors (OPDs) are increasingly paid attention for their broad application prospect. However, it is extremely difficult to achieve highly sensitive detection of polarized light for most organic semiconductor polymers with intrinsic isotropic amorphous behavior. In this study, the performance-enhanced intrinsic anisotropic all-polymer bulk heterojunctions (BHJs) are obtained by constructing molecular fluorination engineering in the fused-ring backbone of the acceptor polymer. The molecular orientation, alignment, and packing can be effectively optimized during the floating-film formation process without using any additives and post-treatments. The outstanding intrinsic polarization-sensitive photodetection performance with a very high photocurrent dichroic ratio of 3.73, a specific detectivity of 1.3 × 1011 Jones at 0 V, and a broad linear dynamic range of 120 dB is achieved for the optimum-fluorine-content self-assembly all-polymer organic BHJ film. The superiorities of self-powered capability, fast response, and high-contrast imaging demonstrate that the backbone fluorination for acceptor polymers is conducive to achieving excellent structural regularity and can endow the intrinsic polarization-sensitive all-polymer OPDs with more demanding polarized-light detection ability.