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Rapid Metabolite-Sensing System for Blood Lactate

Enzyme-driven fluorescent biosensing system rapidly measures the levels of blood lactate and other metabolites, with potential to provide fast diagnostics at the point-of-care

In emergency medicine, blood lactate levels are a reliable real-time indicator of the severity and mortality risk of conditions that occur as a result of poor blood circulation and oxygen supply to organs and tissues (hypoperfusion), such as in patients with sepsis, cardiac arrest, stroke, major trauma, cystic fibrosis and other conditions. Lactate levels also provide an early measure for the patients’ response to treatments. Blood lactate testing in intensive care medicine is typically performed with laboratory analyzers which results in an approximate three-hour delay between triage and blood lactate result due to the need for sample transport to the hospital laboratory. However, this time often significantly exceeds the conceptual “golden hour”, the not clearly defined but relatively short period of time following a hypoperfusion-inducing event during which it is most likely that medical and surgical treatment can prevent death or serious long-term consequences.

To help guide doctors’ decisions, Wyss Institute researchers have created a much more rapid lactate-sensing method that is based on a highly responsive fluorescence biosensing system and provides accurate lactate measurements in a few minutes, making it ideal to be developed for testing at and off the bedside.

Rapid Metabolite-Sensing System for Blood Lactate
This scanning electron cryomicroscopic (CryoSEM) image shows the size and morphology of lactate-sensing liposomes produced by the approach. Credit: Wyss Institute at Harvard University.

Engineering a lactate sensor

In conditions of rampant infection with pathogens, acute cardiovascular insults, or severe physical injury, the blood supply to tissues and organs can be significantly compromised. As a consequence, tissues receive insufficient levels of oxygen, which causes them to switch from oxygen-dependent (aerobic) to oxygen-independent (anaerobic) metabolism to support their energy needs. As anaerobic energy metabolism produces lactate, which eventually enters the central blood circulation, blood lactate levels indirectly report the overall perfusion state of the patient.

By engineering a two-step enzymatic process and encapsulating it into liposomes, Simon Matoori, Ph.D., and David Mooney, Ph.D. created a vesicular reaction compartment that, when added to blood samples, takes up lactate and indicates its levels by a corresponding linear decrease of a fluorescent signal in a clinically relevant lactate range. In the first reaction lactate is converted to the metabolite pyruvate and the reactive oxygen species hydrogen peroxide by the lactase oxidase enzyme encapsulated in the vesicles. Then, in a second chemical reaction, the thus generated hydrogen peroxide is used by another enzyme, horseradish peroxidase (HRP), to oxidize a specifically selected fluorescent dye molecule, causing its fluorescence to be extinguished.

Rapid Metabolite-Sensing System for Blood Lactate
This schematic describes the two-step enzymatic reaction in the liposomal reaction compartment. The metabolite of interest (e.g. lactate; white triangle) diffuses across the membrane and is oxidized by a metabolite-specific enzyme (blue shape) which generates hydrogen peroxide in the process. HRP is then in turn used by hydrogen peroxidase (HRP; green shape) to oxidize the fluorescent dye sulfo-cyanine 7 (red star), extinguishing its near-infrared fluorescence (grey star) and enabling metabolite quantification by fluorometry. Credit: Wyss Institute at Harvard University

Key to this enzymatic strategy was the identification of the fluorescent dye molecule sulfo-cyanine 7 as an HRP substrate that fluoresces in the near-infrared spectrum of light. In the absence of HRP, sulfo-cyanine 7 is highly stable, water-soluble, and emits fluorescence in the near-infrared spectrum, which is important since it cannot be interfered with by hemoglobin present at high concentrations in blood. In addition, by encapsulating the entire enzymatic process in liposome vesicles, the hydrogen peroxide produced in the first enzyme reaction is protected from enzymes in red blood cells that would otherwise rapidly degrade it. Proof-of-concept studies by the research team have demonstrated that the fluorescent lactate assay produces accurate lactate measurements in a clinically relevant range within ten minutes, which is considerably faster than current lactate testing in emergency medicine, which in addition necessitates transport of patients to the hospital.

In the future, these lactate-sensing fluorescent liposomes could be added to a drop of patient blood, and the resulting loss of fluorescence quantified by a small portable fluorometer. The fluorescence signal could then be converted to the corresponding blood lactate level and the results displayed on a smartphone.

Besides its application in emergency medicine, the lactate biosensing system could also be used in sports medicine as athletes evaluate their performance and optimize their training regimen on the basis of blood lactate levels. Lactate is formed when muscles are heavily used and cannot retrieve enough oxygen from the blood circulation, forcing them to shift their energy metabolism from an oxygen-dependent to an oxygen-independent mode. The researchers are currently adapting their method to measuring lactate levels non-invasively in sweat which combined with portable fluorometer could provide athletes with an on-the-go performance indicator.

Versatile metabolite biosensing platform

The stoichiometric conversion of lactate to hydrogen peroxide in the engineered enzymatic reaction compartment, and the quantification of the generated hydrogen peroxide by a fluorescent reporter dye, offered the opportunity to sense other clinically relevant metabolites. By substituting the first lactate-specific enzyme reaction for other reactions that produce hydrogen peroxide in response to other metabolites, such as glucose, ethanol/methanol, and urate, the researchers have created simple and rapid biosensing systems with potential relevance for conditions like diabetes mellitus, alcohol and methanol poisoning, and gout, respectively. The approach thus presents a versatile metabolite biosensing platform, and has potential to rapidly assay levels of a range of metabolites, as well as drugs, in blood and other body fluids, including sweat and urine.

This technology is currently being de-risked at the Wyss Institute and is available for licensing.

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