A portable blood lactate sensor with a non-immobilized enzyme for early sepsis diagnosis

Kiatamornrak, Patcharakorn; Boobphahom, Siraprapa; Lertussavavivat, Tanat; Rattanawaleedirojn, Pranee; Chailapakul, Orawon; Rodthongkum, Nadnudda; Srisawat, Nattachai

doi:10.1039/d2an00218c

Cited by 7 publications

(3 citation statements)

References 37 publications

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“…LOx was used for HRPF benchmarking due to its large size (167 kDa tetramer assembly) and our interests in wearable lactate biosensors for tracking hypoxia in airmen, fatigue monitoring in sports and sepsis in critical care. [33][34][35][36][37][38] "Inoperando" protein footprinting experiments with clinical human samples can help in rational optimization of LOx constructs and sensor design for wearable lactate biosensor development. However, the •OH in HRPF readily react with components of human samples, like glucose, leading to undesirable secondary radical reactions and reduction of the total •OH concentration available for labeling reaction with the LOx enzyme.…”

Section: Methionine Amidementioning

confidence: 99%

Evaluating mass spectrometry based hydroxyl radical protein foot-printing of a benchtop Flash oxidation system against a synchrotron X-ray beamline.

Jain,

Dhillon,

Vijayalakshmi

et al. 2023

Preprint

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Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation and mass spectrometry is a well-validated structural biology method that is providing critical insights into macromolecular dynamics. Numerous alternative sources for HRPF such as laser photolysis and plasma irradiation complement synchrotron-based HRPF. A recently developed commercially available instrument based on flash lamp photolysis, the Fox® system, enables access to laboratory benchtop HRPF. Here, we evaluate the feasibility of standardizing HRPF experiments in-house with a benchtop Fox® instrument as a precursor to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase enzyme (LOx) as a model system, we carried out hydroxyl radical (•OH) labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed with high glucose concentrations to mimic highly scavenging conditions in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the Fox® and XFP HRPF methods was compared, and we found that tuning •OH dosage enabled an optimum labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of Fox® and XFP labeling approaches, showing that benchtop instruments such as Fox® photolysis system can increase throughput and accessibility of HRPF technology.

show abstract

Section: Methionine Amidementioning

confidence: 99%

Evaluating mass spectrometry based hydroxyl radical protein foot-printing of a benchtop Flash oxidation system against a synchrotron X-ray beamline.

Jain,

Dhillon,

Vijayalakshmi

et al. 2023

Preprint

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show abstract

“…We have benchmarked the •OH labeling of FOX against a synchrotron XFP beamline using lactate oxidase (LOx) as a model protein system. LOx was used for HRPF benchmarking due to its large size (167 kDa tetramer assembly) and our interests in wearable lactate biosensors for tracking hypoxia in airmen, fatigue monitoring in sports, and sepsis in critical care. − “ In operando ” protein footprinting experiments with clinical samples can help in the rational optimization of LOx constructs and sensor design for wearable lactate biosensor development. However, the •OH in HRPF on clinical samples may readily react with components of human plasma, like vital proteins (such as albumin) and metabolites (such as sugars and fats), leading to undesirable secondary radical reactions and reduction of the total •OH concentration available for the labeling reaction of the LOx enzyme .…”

Section: Introductionmentioning

confidence: 99%

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline

Jain,

Dhillon,

Kanchustambham

et al. 2024

J. Am. Soc. Mass Spectrom.

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Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

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“…In addition, ABG can also cause a high-risk infection when the blood sample is frequently collected from an artery [ 10 ]. To overcome these issues, electrochemical biosensors are being explored due to their high sensitivity, selectivity, simplicity, rapid response, cost-effectiveness, lack of requirement for sample preparation, and the considerable ease in developing a portable device for point-of-care (POC) diagnosis [ 11 , 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

Disposable Polyaniline/m-Phenylenediamine-Based Electrochemical Lactate Biosensor for Early Sepsis Diagnosis

Thongkhao,

Numnuam,

Khongkow

et al. 2024

Polymers

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Lactate serves as a crucial biomarker that indicates sepsis assessment in critically ill patients. A rapid, accurate, and portable analytical device for lactate detection is required. This work developed a stepwise polyurethane–polyaniline–m-phenylenediamine via a layer-by-layer based electrochemical biosensor, using a screen-printed gold electrode for lactate determination in blood samples. The developed lactate biosensor was electrochemically fabricated with layers of m-phenylenediamine, polyaniline, a crosslinking of a small amount of lactate oxidase via glutaraldehyde, and polyurethane as an outer membrane. The lactate determination using amperometry revealed the biosensor’s performance with a wide linear range of 0.20–5.0 mmol L−1, a sensitivity of 12.17 ± 0.02 µA·mmol−1·L·cm−2, and a detection limit of 7.9 µmol L−1. The developed biosensor exhibited a fast response time of 5 s, high selectivity, excellent long-term storage stability over 10 weeks, and good reproducibility with 3.74% RSD. Additionally, the determination of lactate in human blood plasma using the developed lactate biosensor was examined. The results were in agreement with the enzymatic colorimetric gold standard method (p > 0.05). Our developed biosensor provides efficiency, reliability, and is a great potential tool for advancing lactate point-of-care testing applications in the early diagnosis of sepsis.

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A portable blood lactate sensor with a non-immobilized enzyme for early sepsis diagnosis

Abstract: Early determination of blood lactate levels may accelerate the detection of sepsis, one of the most time-sensitive illnesses.

Cited by 7 publications

References 37 publications

Evaluating mass spectrometry based hydroxyl radical protein foot-printing of a benchtop Flash oxidation system against a synchrotron X-ray beamline.

Evaluating mass spectrometry based hydroxyl radical protein foot-printing of a benchtop Flash oxidation system against a synchrotron X-ray beamline.

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline

Disposable Polyaniline/m-Phenylenediamine-Based Electrochemical Lactate Biosensor for Early Sepsis Diagnosis

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