SLAS Technology

Real-time reverse transcription polymerase chain reaction (rRT-PCR) is the primary method used for the detection and diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since SARS-CoV-2s entrance into the United States, numerous clinical laboratories and in vitro diagnostic companies have developed rRT-PCR assays, some requiring specialized materials and reagents. One such assay includes the cobas(R) SARS-CoV-2 Qualitative Assay for use on the cobas(R) 6800/8800 (Roche Molecular Systems, Inc.). Since initiation of this assay at our facility, our ability to run testing at full capacity has been limited due to restricted supply of the omni cobas(R) Processing Plate (Product Number 05534917001), a 96 deep well plate used for all sample processing and total nucleic acid extraction via MagNA Pure magnetic beads. To work around this limiting factor, we have successfully designed and tested a cleaning protocol utilizing the widely available laboratory resources of bleach, ethyl-alcohol and autoclaving, for omni cobas(R) Processing Plate re-use.

When testing large numbers of clinical COVID-19 samples for diagnostic purposes, pooling samples together for processing can offer significant reductions in the materials, reagents, time, and labor needed. We have evaluated two different strategies for pooling independent nasopharyngeal swab samples prior to testing with an EUA-approved SARS-CoV-2 RT-qPCR diagnostic assay. First, in the Dilution Study, we assessed the assay's ability to detect a single positive clinical sample diluted in multiple negative samples before the viral RNA extraction stage. We observed that positive samples with Ct values at ~30 can be reliably detected in pools of up to 30 independent samples, and positive samples with Ct values at ~35 can be detected in pools of 5 samples. Second, in the Reloading Study, we assessed the efficacy of reloading QIAamp viral RNA extraction columns numerous times using a single positive sample and multiple negative samples. We determined that one RNA extraction column can be reloaded with up to 20 clinical samples (1 positive and 19 negatives) sequentially without any loss of signal in the diagnostic assay. Furthermore, we found there was no significant difference in assay readout whether the positive sample was loaded first or last in a series of 20 samples. These results demonstrate that different pooling strategies can lead to increased process efficiencies for COVID-19 clinical diagnostic testing.

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

Human proteases play major roles in various pathological conditions, including dysregulated immune responses in sepsis, making them strong candidates for developing diagnostic markers. Despite this potential, the progress of developing protease-based diagnostic tools has remained slow due to significant technical barriers associated with measuring protease activity, mainly stemming from the vast diversity and the lack of substrate specificity, which complicate the interpretation of protease activity profiles. In this work, we advanced the current state of assay development by designing substrate molecule sensors and implementing an analytical approach based on mass spectrometry. Specifically, we chemically modified protease substrates for human neutrophil elastase (HNE) and matrix metalloproteinases (MMPs) to enhance specificity in mass spectrometry. This approach yields distinct cleavage products with non-overlapping mass-to-charge signatures, allowing precise differentiation of each proteases activity. We then integrated the modified substrates into a mass spectrometry-based multiplexed assay platform, enabling quantification of multiple protease activities in a single run. We applied the assay to plasma samples and demonstrated that the assay detects distinct protease activity profiles. Our study demonstrated that the assay achieved a diagnostic sensitivity of 88% and specificity of 87% for sepsis detection. The combination of low cost, rapidness, and robust diagnostic performance makes this platform well-suited to a wide range of clinical settings. One Sentence SummaryNovel modifications to protease substrates enable a multiplexed activity assay for accurate sepsis diagnosis in a 3-hour timeframe.

Stable and productive CHO cell lines are essential for biopharmaceutical manufacturing, yet early expansion steps are often constrained by prolonged period required for suspension adaptation. Single-cell cloning (SCC) ensures monoclonality and regulatory compliance, but cells transitioning from static to suspension culture frequently exhibit variable recovery, which prolongs timelines and increases process variability. To address this challenge, mixing-based microplate culture systems have been developed to improve early expansion efficiency. The C.NEST platform provides controlled pneumatic mixing and environmental monitoring that facilitates earlier adaptation to suspension conditions. At the 96-well and 24-well stages, this system allows cells to establish stable growth under suspension-like environments, thereby shortening the adaptation period following transfer to shaking culture. In this study, we applied C.NEST to the SCC workflow for developing CHO-K1 stable cell lines. Integrating C.NESTs controlled mixing reduced adaptation time, enhanced the consistency of clone expansion, and improved the ability to identify high-yield clones. These findings highlight the potential of C.NEST to streamline cell line development workflows by accelerating early suspension adaptation and improving clone selection reliability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/693844v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ca76bforg.highwire.dtl.DTLVardef@3a3079org.highwire.dtl.DTLVardef@447e01org.highwire.dtl.DTLVardef@ac8d6f_HPS_FORMAT_FIGEXP M_FIG C_FIG C.NEST mixing shortens suspension adaptation, accelerates clone expansion, and enhances early-stage screening. HighlightO_LIThe C.NEST microplate agitation culture system accelerates early CHO-K1 cell line development. C_LIO_LIControlled pneumatic mixing improved oxygen transfer and medium homogeneity, promoting stable growth during early expansion. C_LIO_LIEarly mixing shortens suspension adaptation by approximately one week. C_LIO_LIMixing cultures enabled more accurate clone performance assessment, revealing high-producing outliers. C_LIO_LIC.NEST provides a scalable and reproducible solution for integrating mixing-based culture into single-cell cloning workflows. C_LI

The primary goal in cell line development is to establish high-producer recombinant cell line(s) of single-cell origin. Traditionally, these cell lines are developed using limiting dilution cloning (LDC) method of single cell isolation, a rate-limiting, lengthy and labor-intensive process. The Verified-In-Situ-Plate-Seeding (VIPS) is an automated single-cell seeding and imaging equipment designed to accelerate cell line development workflow. In this study, VIPS was tested for efficiency and accuracy of single cell seeding in parallel with limiting dilution cloning (LDC). Three Chinese hamster ovary (CHO) derived cell lines with known clonal properties were tested under six different growth conditions (three growth media and two different kinds of microplates). Data showed VIPS and limiting dilution (LDC) have comparable cloning efficiency when CHO-M cells were tested. By contrast, the Verified In-Situ Plate Seeding (VIPS) produced 6-8-fold more clones of single-cell origin than LDC when CHO-K1 or CHO-S cells were tested. Moreover, the verified In-Situ Plate Seeding (VIPS) correctly identified single-cell and multiple-cells seeded wells with 65-72% and 52-81% accuracy, respectively. Taken together, the high throughput imaging and single-cell seeding capabilities of VIPS outperformed the rate-limiting LDC method and, therefore, has the potential to accelerate cell line development workflow.

Sepsis is the bodys dysfunctional response to infection associated with organ failure. Delays in diagnosis have a substantial impact on survival. Herein, samples from 586 in-house patients were used in conjunction with machine learning and cross-validation to narrow a gene expression signature of immune cell reprogramming to predict clinical deterioration in patients with suspected sepsis within the first 24 hours (h) of clinical presentation using just six genes (Sepset). The accuracy of the test ([~]90% in early intensive care unit (ICU) and 70% in emergency room patients) was validated in 3,178 patients from existing independent cohorts. A real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based test was shown to have a 98% sensitivity in >230 patients to predict worsening of the sequential organ failure scores or admission to the ICU within the first 24 h following Sepset detection. A stand-alone centrifugal microfluidic instrument that integrates the entire automated workflow for detection of the Sepset classifier in whole blood using digital droplet PCR was developed and tested. This PREcision meDIcine for CriTical care (PREDICT) system had a high sensitivity of 92%, specificity of 89%, and an overall accuracy of 88% in identifying the risk of imminent clinical deterioration in patients with suspected sepsis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/24314844v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@82f577org.highwire.dtl.DTLVardef@1c18921org.highwire.dtl.DTLVardef@111f119org.highwire.dtl.DTLVardef@ebbb87_HPS_FORMAT_FIGEXP M_FIG Description of Graphic AbstractFeature reduction and development of a gene classifier that predicts deterioration-risk-groups in patients starts with in-house RNA sequencing data from patient collected from a heterogenous cohort of patients with suspected sepsis (top left) to reduce our original published gene signature down to 6-genes (Sepset), for which expression could be related to 2 housekeeping genes. Feature selection was performed using machine learning (ML) and AI and the classifier validated in samples from published transcriptomic studies. Molecular assay is then developed by designing and testing primer/probe sequences specific to the target genes using digital droplet PCR. In parallel, sample-to-answer microfluidic platform and cartridges are developed (bottom right) and analytical performance of multiplex quantitative assay is tested. Prognostic enrichment is obtained by analyzing the results using ML algorithm to determine the percent likelihood of significant clinical deterioration within the immediate next 24 h. The deployment of PREDICT platform (center) at the point-of-care is anticipated to aid in triage and management of prospective sepsis within the first 3 h of clinical presentation. C_FIG

To advance the industrialization of cultured meat and regenerative medicine, scalable and efficient cell culture techniques are essential. Among these, the suspension culture method using microcarriers has emerged as a promising approach for the large-scale cell culture technique. However, monitoring cell growth on the microcarriers remains challenging, particularly in developing cell counting techniques that can be seamlessly integrated into bioprocess workflows without cell detachment, fluorescence labeling and any parameter tuning in the analysis algorithm. In this study, we proposed a versatile image analysis-based cell counting method by using cellular autofluorescence without any parameter tuning. The proposed method estimates the number of cells by applying spatiotemporal averaging to the autofluorescence signals in the microscopic images. Using numerical and cell culture experiments, we demonstrated that the proposed method can estimates the number of cells accurately. This technique, which harnesses the ubiquitous autofluorescence inherent in living cells, offers a cost-effective and practical solution applicable to a broad range of fields requiring high-throughput cell quantification.

Seasonal flu and pandemics, which account for millions of infections and hundreds of thousands of deaths, require rapid and reliable detection mechanisms to implement preventive and therapeutic measures. Current detection methods of viral infections have limitations in speed, accuracy, accessibility, and usability. This project presents a novel, widely applicable viral diagnostic test that uses a modified version of rolling circle amplification (RCA) to be sensitive, specific, direct RNA targeted, colorimetric and operable at room temperature. We are specifically detecting the following high-impact viruses: SARS-CoV-2, Influenza A (H1N1pdm09), and Influenza B (Victoria Lineage), although our test can be adapted to any viral infection. Results using synthetic viral DNA and RNA sequences show that our diagnostic test takes approximately one hour, detects femtomolar concentrations of RNA strands, and differentiates between virus strains. We believe implementing our diagnostic test will provide faster responses to future viral-related outbreaks for quicker societal recovery.

New in vitro models are an urgent need for to improve both the research data and the preclinical development of new drugs. Current standardized cellular models are mainly based in 2D cell culture, which lacks flow conditions and with complex co-culture settings. In this way, advanced cell culture models, such as organ-on-a-chip (OoC), aim to solve these limitations. OoC systems are composed by a microfluidic chip functionalized with different combinations of extracellular matrixes, coatings and cell cultures to mimic the physiological conditions of human organs. Advantages of OoC include the possibility to add 3D structures, delimited regions for co-culture and dynamic flow conditions to cell cultures. However, to perform reproducible and controlled experiments with OoC, it is necessary to systematically standardize the cell culture conditions in the microfluidic channels. For this is necessary to test both the combination of flow and extracellular matrix (ECM) coating to reliably mimic the human organ physiology. In this work, we standardized both conditions, ECM coating and the flow conditions to functionalize OoC with cell lines from kidney (Caki-1) and from lung (A549) to develop OoC systems beyond the Vessel-on-a-chip setting. In this way, the protocol detailed in this work will allow to standardize cell culture on different optimized OoC types with different cell types from different origins.

Biomedicine today is experiencing a shift towards decentralized data collection, which promises enhanced reproducibility and collaboration across diverse laboratory environments. This inter-laboratory study evaluates the performance of biocytometry, a method utilizing engineered bioparticles for enumerating cells based on their surface antigen patterns. In a decentralized framework, spanning 78 assays conducted by 30 users across 12 distinct laboratories, biocytometry consistently demonstrated significant statistical power in discriminating numbers of target cells at varying concentrations as low as 1 cell per 100,000 background cells. User skill levels varied from expert to beginner capturing a range of proficiencies. Measurement was performed in a decentralized environment without any instrument cross-calibration or advanced user training outside of a basic instruction manual. The results affirm biocytometry to be a viable solution for immunophenotyping applications demanding sensitivity as well as scalability and reproducibility and paves the way for decentralized analysis of rare cells in heterogeneous samples.

Live imaging of human or other mammalian cells at multi-hour time scales with minimal perturbation to their growth state requires that the specimens optimal growth conditions are met while fixed to a microscope stage. In general, ideal conditions include culturing in complete growth media, an ambient temperature of 36-37 C, and a humidity-controlled atmosphere comprising typically 5-7% CO2. Commercially available devices that achieve these conditions are not a financially viable option for many labs, with the price ranging anywhere from $12000 to $40000. The advent of 3D printing technology has allowed for low-cost rapid prototyping with precision comparable to traditional fabrication methods, opening the possibility for in-lab design and production of otherwise prohibitively expensive equipment such as stage-top incubation devices. The continued usefulness and widespread availability of single-board computers (SBC) such as Arduino and Raspberry Pi also simplify the process by which these devices can be controlled. Here we report the production of a do-it-yourself (DIY) device for stage-top incubation with temperature and atmospheric control with a cost reduction of approximately 100x.

Deaths from coronavirus disease (COVID-19) have exceeded 300,000 persons globally, calling for rapid development of mobile diagnostics that can assay widespread prevalence and infection rates. Data provided in this study supports the utility of a newly-designed lateral flow immunoassay (LFA) for detecting SARS-CoV-2 IgM and IgG antibodies. We employed a clinical cohort of 1,892 SARS-CoV-2 patients and controls, including individuals diagnosed by RT-qPCR at Yale New Haven Hospital, The First Affiliated Hospital of Anhui Medical University, the Chinese Center for Disease Control and Prevention of Hefei City (Hefei CDC), Anhui Province (Anhui Province CDC), and Fuyang City (Fuyang CDC). The LFA studied here detects SARS-CoV-2 IgM and IgG antibodies with a specificity of 97.9-100% for IgM, 99.7-100% for IgG, and sensitivities ranging from 94.1-100% for patients >14-days post symptom onset. Sensitivity decreases in patients <14-days post symptom onset, which is likely due to lower IgG/IgM antibody levels in this population. Finally, we developed a visual intensity reporting system that we believe will be suitable for laboratory and point-of-care settings, and will provide granular information about antibody levels. Overall our results support the widespread utility of this and other LFAs in assessing population-level epidemiological statistics.

As the United States prepares to return to work and open up the economy in the midst of the COVID-19 pandemic without an available vaccine or effective therapy, testing and contact tracing are essential to contain and limit the spread of the COVID-19 virus. In response to the urgent public health need for accurate, effective, low-cost, and scalable COVID-19 testing technology, we evaluated and identified diagnostic solutions with potential for use as an at-home product. We conducted a deep horizon scan for antigen and serology-based diagnostics and down-selected to the most promising technologies. A total of 303 candidate products (138 antibody and 44 antigen tests) were identified. Product evaluations were based entirely on company-provided data. 73 serology-based antibody tests passing an initial scoring algorithm based on specificity and sensitivity data were then further evaluated using a second scoring algorithm. This second algorithm included a review of additional technical specifications of the devices, an analysis of supply chain, manufacturing, and distribution capacity of each vendor. 24 potential antibody products met the selection criteria for further direct laboratory evaluation. The performance metrics for selection of these 24 products are currently being evaluated in a Mass General Brigham laboratory. Testing alone might not be sufficient to prevent the spread of a highly contagious disease like COVID-19. Manual contact tracing could complement testing, but it is likely to fail in identifying many individuals who were in contact with a given COVID patient. The proliferation of smartphones in the population has enabled the development of solutions that can provide public health officials with valuable information for rapid and accurate contact tracing. Besides, electronic-based contact tracing solutions can be augmented by symptom self-reports gathered using electronic patient reported outcome (ePRO) platforms and by physiological data collected using wearable sensors. We performed a detailed assessment of 12 ePRO solutions, 27 wearable sensors, and 44 electronic-based contact tracing solutions. These technologies were evaluated using criteria developed to assess their suitability to address the COVID-19 pandemic. We identified a number of solutions that could augment if not provide a more effective alternative to manual contact tracing. Finally, we propose a theoretical framework in which ePRO platforms, wearable sensors, and electronic-based contact tracing solutions would be utilized in combination with molecular and serological tests to identify and isolate COVID-19 cases rapidly.

The recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times.

Extracellular adenosine triphosphate (eATP) is a mediator of purinergic signalling in the airways, implicated in mucociliary function, inflammation, and cough via activation of P2X3 receptors. Elevated airway eATP has been associated with multiple respiratory diseases, yet reliable measurement of eATP remains challenging due to its rapid enzymatic degradation and confounding contributions from intracellular ATP. Here, we describe an optimized, microwell plate-based luminescence method for quantifying eATP from human airway epithelial cell cultures and bronchoalveolar lavage (BAL) fluid with enhanced signal stability. Using a commercially available ATP detection assay with a prolonged luminescence half-life, we introduced a simple 0.45 {micro}m syringe filtration step to remove cells and thereby isolate extracellular ATP. This approach demonstrated ATP specificity via apyrase degradation, and provided a linear detection range from 5 nM to 5 {micro}M. Addition of ATP stabilization buffer preserved ATP levels in cell culture media for at least 4 hours at 4 {degrees}C and in human BAL samples for at least 6 weeks at -80{degrees}C. Applying this method to primary human bronchial epithelial cells revealed detectable eATP release, with preferential secretion at the apical surface under air-liquid interface conditions. Collectively, this optimized assay enables robust, high-throughput, and time-flexible quantification of eATP in both experimental and clinical airway samples. These methods support improved investigation of purinergic signalling in airway health and disease and may facilitate biomarker development relevant to eATP in the airways.

Scratch assays are routinely performed for multiple research applications in cell biology (i.e., migration, metastasis, repair mechanisms, etc.). Conventional scratches are usually generated with pipette tips. User inconsistencies and varying pipette tips are major obstacles to reproducibility and quantitative power. Here, we present a novel, scratch/gap generating, cell culture plate-based, platform that addresses these issues, providing consistency and reproducibility across multiple users, significantly reducing variability, and increasing quantitative outcomes.

Drug testing is a vital step in identification of the potential efficacy of any new/existing drug and/or combinations of drugs. The conventional methods of testing the efficacy of new drugs using multi-well plates are time consuming, prone to evaporation loss and manual error. Microfluidic devices with automated generation of concentration gradient provide a promising alternative. The implementation of such microfluidic devices is still limited owing to the additional expertise and facilities required to fabricate and run these devices. Conventional microfluidic devices also need pumps, tubings, valves, and other accessories, making them bulky and nonportable. To address these problems, we have developed a method for fabricating microfluidic structures using a nonconventional technique by exploiting the Saffman-Taylor instability in lifted Hele-Shaw cell. Multi-channel structure molds with varying dimensions were fabricated by shaping ceramic polymer slurry and retaining the shape. Further using the mold thus made, polydimethyl siloxane (PDMS) devices offering static, stable, diffusion-based gradient were casted using soft lithography. We have demonstrated with COMSOL simulation, as well as using Fluorescein isothiocyanate (FITC), a fluorescent dye, that the concentration gradient can be generated in this device, which remains stable for at least 5 days. Using this multichannel device, in vitro drug efficacy was validated with two drugs namely-Temozolomide (TMZ) and Curcumin, one FDA approved and one under research, on glioblastoma cells (U87MG). The resulting IC50 values were consistent with those reported in literature. We have also demonstrated the possibility of conducting molecular assays post-drug testing in the device by microtubule staining after curcumin treatment on cervical cancer cells (HeLa). In summary, we have demonstrated a i) user-friendly, ii) portable, static drug testing platform that iii) does not require further accessories and can create iv) a stable gradient for long duration. Such a device can reduce the time, manual errors, fabrication and running expenditure, and resources to a great extent in drug testing. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/505813v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@e1f648org.highwire.dtl.DTLVardef@f029d7org.highwire.dtl.DTLVardef@14afd68org.highwire.dtl.DTLVardef@42b888_HPS_FORMAT_FIGEXP M_FIG C_FIG

Metabolism is essential for cellular functions and is often altered in cancer. While Seahorse assays are well established for measuring metabolic changes in 2D cell cultures, their application to 3D models remains challenging. Here, we present a step-by-step protocol for plating individual brain tumor neurospheres in the assay microplate to measure their bioenergetics. We provide actionable recommendations and highlight pitfalls to avoid. This approach enables exploration of dynamic metabolic changes in patient-derived brain tumor neurospheres following genetic or pharmacologic interventions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/691986v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@25cb34org.highwire.dtl.DTLVardef@13ff857org.highwire.dtl.DTLVardef@c91c7forg.highwire.dtl.DTLVardef@16dfa27_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIOptimization of Seahorse assay protocol for single patient-derived brain tumor spheroids C_LIO_LIStep-by-step instructions for successfully re-plating and transferring single brain tumor spheroids C_LIO_LIImportance of microplate coating for the successful execution of Seahorse assays C_LIO_LIImaging of the microplate before and after the assay allows for normalization of metabolic parameters based on neurosphere size C_LI

Lower Respiratory Tract Infections (LRTIs) represent the leading cause of death due to infectious diseases. Current diagnostic modalities primarily depend on clinical symptoms and lack specificity, especially in light of common colonization without overt infection. To address this, we developed a noninvasive diagnostic approach that employs BreathBiomics, an advanced human breath sampling system, to detect protease activities induced by bacterial infection in the lower respiratory tract. Specifically, we engineered a high-sensitivity and high-specificity molecular sensor for human neutrophil elastase (HNE). The sensor undergoes cleavage in the presence of HNE, an event that is subsequently detected via Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Application of this methodology to clinical samples, breath specimens collected from intubated patients with LRTIs, demonstrated the detection of the cleaved sensor by MALDI-TOF MS. Our findings indicate that this novel approach offers a noninvasive and specific diagnostic strategy for people with LRTIs. O_TEXTBOXSignificance The potential for using human breath for noninvasive disease detection and diagnosis has long been recognized, yet the lack of effective biomolecular sampling technologies has hindered progress. To address this limitation, we developed BreathBiomics, an advanced sampling system designed to efficiently capture biomolecules in human exhaled breath. By focusing on protease dysregulation, an established event induced by bacterial infections, we demonstrated that BreathBiomics can capture proteases and facilitate their subsequent activity-based detection for the diagnosis of LRTI. We verified the assays sensitivity and clinical applicability through empirical studies. Our work marks a significant advancement by providing the first viable pathway for the development of in vitro diagnostic assays leveraging human breath for disease detection and diagnosis. C_TEXTBOX