Exploring microplastic impact on whole blood clotting dynamics utilizing thromboelastography.

This study investigates the influence of microplastics on blood clotting. It addresses the lack of comprehensive research on the effects of microplastic size and surface modification on clotting dynamics in human whole blood. Thromboelastography was used to examine aminated (aPS), carboxylated (cPS), and non-functionalized (nPS) polystyrene particles with sizes of 50, 100, and 500 nm. Results show that cPS consistently activated the clotting cascade, demonstrating increased fibrin polymerization rates, and enhanced clot strength in a size and concentration-dependent manner. nPS had minimal effects on clotting dynamics except for 50 nm particles at the lowest concentration. The clotting effects of aPS (100 nm particles) resembled those of cPS but were diminished in the 500 nm aPS group. These findings emphasize the importance of microplastic surface modification, size, concentration, and surface area on in-vitro whole blood clotting dynamics.

Effect of Chandler loop shear and tubing size on thrombus architecture.

Thrombosis can lead to a wide variety of life-threatening circumstances. As current thrombolytic drug screening models often poorly predict drug profiles, leading to failure of thrombolytic therapy or clinical translation, more representative clot substrates are necessary for drug evaluation. Utilizing a Chandler loop device to form clot analogs at high shear has gained popularity in stroke societies. However, shear-dependent clot microstructure has not been fully addressed and low shear conditions are often overlooked. We herein characterized the impact of wall shear rate (126 to 951 s-1) on clot properties in the Chandler loop. Different revolutions (20-60) per minute and tubing sizes (3.2 to 7.9 mm) were employed to create different sized clots to mimic various thrombosis applications. Increased shear resulted in decreased RBC counts (76.9 ± 4.3% to 17.6 ± 0.9%) and increased fibrin (10 to 60%) based on clot histology. Increased fibrin sheet morphology and platelet aggregates were observed at higher shear under scanning electron microscope. These results show the significant impact of shear and tubing size on resulting clot properties and demonstrate the capability of forming a variety of reproducible in-vivo-like clot analogs in the Chandler loop device controlling for simple parameters to tune clot characteristics.

Real-time tracking of fibrinolysis under constant wall shear and various pulsatile flows in an in-vitro thrombolysis model.

A great need exists for the development of a more representative in-vitro model to efficiently screen novel thrombolytic therapies. We herein report the design, validation, and characterization of a highly reproducible, physiological scale, flowing clot lysis platform with real-time fibrinolysis monitoring to screen thrombolytic drugs utilizing a fluorescein isothiocyanate (FITC)-labeled clot analog. Using this Real-Time Fluorometric Flowing Fibrinolysis assay (RT-FluFF assay), a tPa-dependent degree of thrombolysis was observed both via clot mass loss as well as fluorometrically monitored release of FITC-labeled fibrin degradation products. Percent clot mass loss ranged from 33.6% to 85.9% with fluorescence release rates of 0.53 to 1.17 RFU/min in 40 and 1000 ng/mL tPa conditions, respectively. The platform is easily adapted to produce pulsatile flows. Hemodynamics of human main pulmonary artery were mimicked through matching dimensionless flow parameters calculated using clinical data. Increasing pressure amplitude range (4-40 mmHg) results in a 20% increase of fibrinolysis at 1000 ng/mL tPA. Increasing shear flow rate (205-913 s-1) significantly increases fibrinolysis and mechanical digestion. These findings suggest pulsatile level affects thrombolytic drug activities and the proposed in-vitro clot model offers a versatile testing platform for thrombolytic drug screening.

Microplastic Effects on Thrombin-Fibrinogen Clotting Dynamics Measured via Turbidity and Thromboelastography.

Micro/nanoplastics, whether manufactured or resulting from environmental degradation, can enter the body through ingestion, inhalation, or dermal pathways. Previous research has found that nanoplastics with diameters of ≤100 nm can translocate into the circulatory system in a dose-dependent manner and potentially impact thrombosis and hemostasis. To investigate the direct effects of microplastics on fibrin clot formation, a simplified ex vivo human thrombin/fibrinogen clot model was utilized. The 100 nm polystyrene particles (non-functionalized [nPS] and aminated [aPS]) were preincubated (0-200 µg/mL) with either thrombin or fibrinogen, and fibrin clot formation was characterized via turbidity and thromboelastography (TEG). When the particles were preincubated with fibrinogen, little effect was observed for aPS or nPS on turbidity or TEG up through 100 µg/mL. TEG results demonstrated a significant impact on clot formation rate and strength, in the case of nPS preincubated with thrombin exhibiting a significant dose-dependent inhibitory effect. In conclusion, the presence of microplastics can have inhibitory effects on fibrin clot formation that are dependent upon both particle surface charge and concentration. Negatively charged nPS exhibited the most significant impacts to clot strength, turbidity, and rate of fibrin formation when first incubated with thrombin, with its impact being greatly diminished when preincubated with fibrinogen in this simplified fibrin clot model.

Multivalent Benzamidine Molecules for Plasmin Inhibition: Effect of Valency and Linker Length.

There is an emerging interest in utilizing synthetic multivalent inhibitors that comprise of multiple inhibitor moieties linked on a common scaffold to achieve strong and selective enzyme inhibition. As multivalent inhibition is impacted by valency and linker length, in this study, we explore the effect of multivalent benzamidine inhibitors of varying valency and linker length on plasmin inhibition. Plasmin is an endogenous enzyme responsible for digesting fibrin present in blood clots. Monovalent plasmin(ogen) inhibitors are utilized clinically to treat hyperfibrinolysis-associated bleeding events. Benzamidine is a reversible inhibitor that binds to plasmin's active site. Herein, multivalent benzamidine inhibitors of varying valencies (mono-, bi- and tri-valent) and linker lengths (∼1-12 nm) were synthesized to systematically study their effect on plasmin inhibition. Inhibition assays were performed using a plasmin substrate (S-2251) to determine inhibition constants (Ki). Pentamidine (shortest bivalent) and Tri-AMB (shortest trivalent) were the strongest inhibitors with Ki values of 2.1±0.8 and 3.9±1.7 µM, respectively. Overall, increasing valency and decreasing linker length, increases effective local concentration of the inhibitor and therefore, resulted in stronger inhibition of plasmin via statistical rebinding. This study aids in the design of multivalent inhibitors that can achieve desired enzyme inhibition by means of modulating valency and linker length.

Stabilization protects islet integrity during respirometry in the Oroboros Oxygraph-2K analyzer.

Metabolic dysfunction of ß-cells has been implicated as a contributor to diabetes pathogenesis, and efforts are ongoing to optimize analytical techniques that evaluate islet metabolism. High-resolution respirometry offers sensitive measurements of the respiratory effects of metabolic substrates and customizable manipulation of electron transport chain components, though the delicate nature of islets can pose challenges to conventional analyses. An affordable and reliable option for respirometry is the Oroboros Oxygraph-2 K system, which utilizes a stir bar to circulate reagents around cells. While this technique may be suitable for individual cells or mitochondria, the continual force exerted by the stir bar can have damaging effects on islet integrity. Herein, we demonstrate the protective benefits of a novel 3D-printed islet stabilization device and highlight the destructive effects of conventional Oxygraph analysis on islet integrity. Islet containment did not inhibit cellular responses to metabolic modulatory drugs, as indicated by robust fluctuations in oxygen consumption rates. The average size of wild-type mouse islets was significantly reduced following a standard Mito Stress Test within Oxygraph chambers, with a clear disruption in islet morphology and viability. Alternatively, containment of the islets within the interior chamber of the islet stabilization device yielded preservation of both islet morphology and increased cell viability/survival after respirometry analysis. Collectively, our study introduces a new and easily accessible tool to improve conventional Oxygraph respirometry of pancreatic islets by preserving natural islet structure and function throughout metabolic analysis.

Use of the "Future Life Map" exercise to improve awareness of career options and opportunities in underrepresented minority undergraduate students pursuing STEM careers.

OBJECTIVES: There has long existed significant underrepresentation of minority students in STEM training and careers. Ongoing efforts to improve opportunities and participation for underrepresented minority students have focused on multiple areas, from increased funding to early exposure to research in STEM. We developed the novel Future Life Map career planning exercise with the goal of contributing to this multi-faceted approach. The exercise emphasizes on the consideration of multiple potential career destinations and routes to those destination. The exercise was designed with the goal of improving participant awareness of options and career planning self-efficacy to improve success and retention of underrepresented minority student participation and retention in STEM. METHODS: We implemented the Future Life Map exercise with 2 separate groups of under-represented minority undergraduate students pursuing careers in STEM. Participants then completed an anonymous survey to evaluate the exercise and describe the value they derived from completing the Future Life Map. RESULTS: The exercise presentation and its supporting documents were highly rated by participants with >81% of respondents rating it as "very informative" (4 or 5 on a 5-point Likert Scale). Participants reported that they were very likely to recommend the exercise to others (25 of 27 participants) and were likely to repeat the activity for their own future decision making (22 participants). Themes that emerged from participant reporting of the value of the exercise were: increased awareness of career and training options, improved understanding of the research required to make informed career/life decisions, and new awareness of specific information about career options under consideration. CONCLUSION: The Future Life Map exercise was successful in improving participant awareness of career options, career planning ability, and helped participants to feel more empowered. This is likely of particular benefit for improving participation and retention of under-represented minority students pursuing careers in STEM.

Modulation of soluble guanylate cyclase ameliorates pulmonary hypertension in a rat model of chronic thromboembolic pulmonary hypertension by stimulating angiogenesis.

Acute pulmonary embolism (PE) does not always resolve after treatment and can progress to chronic thromboembolic disease (CTED) or the more severe chronic thromboembolic pulmonary hypertension (CTEPH). The mechanisms surrounding the likelihood of PE resolution or progress to CTED/CTEPH remain largely unknown. We have developed a rat model of CTEPH that closely resembles the human disease in terms of hemodynamics and cardiac manifestations. Embolization of rats with polystyrene microspheres followed by suppression of angiogenesis with the inhibitor of vascular endothelial growth factor receptor 2 (VEGF-R2) SU5416 results in transient, acute pulmonary hypertension that progresses into chronic PE with PH with sustained right ventricular systolic pressures exceeding 70 mmHg (chronic pulmonary embolism [CPE] model). This model is similar to the widely utilized hypoxia/SU5416 model with the exception that the "first hit" is PE. Rats with CPE have impaired right heart function characterized by reduced VO2 Max, reduced cardiac output, and increased Fulton index. None of these metrics are adversely affected by PE alone. Contrast-mediated CT imaging of lungs from rats with PE minus SU5416 show large increases in pulmonary vascular volume, presumably due to an angiogenic response to acute PE/PH. Co-treatment with SU5416 suppresses angiogenesis and produces the CTEPH-like phenotype. We report here that treatment of CPE rats with agonists for soluble guanylate cyclase, a source of cGMP which is in turn a signal for angiogenesis, markedly increases angiogenesis in lungs, and ameliorates the cardiac deficiencies in the CPE model. These results have implications for future development of therapies for human CTEPH.

Fluorescently conjugated annular fibrin clot for multiplexed real-time digestion analysis.

Impaired fibrinolysis has long been considered as a risk factor for venous thromboembolism. Fibrin clots formed at physiological concentrations are promising substrates for monitoring fibrinolytic performance as they offer clot microstructures resembling in vivo. Here we introduce a fluorescently labeled fibrin clot lysis assay which leverages a unique annular clot geometry assayed using a microplate reader. A physiologically relevant fibrin clotting formulation was explored to achieve high assay sensitivity while minimizing labeling impact as fluorescence isothiocyanate (FITC)-fibrin(ogen) conjugations significantly affect both fibrin polymerization and fibrinolysis. Clot characteristics were examined using thromboelastography (TEG), turbidity, scanning electron microscopy, and confocal microscopy. Sample fibrinolytic activities at varying plasmin, plasminogen, and tissue plasminogen activator (tPA) concentrations were assessed in the present study and results were compared to an S2251 chromogenic assay. The optimized physiologically relevant clot substrate showed minimal reporter-conjugation impact with nearly physiological clot properties. The assay demonstrated good reproducibility, wide working range, kinetic read ability, low limit of detection, and the capability to distinguish fibrin binding-related lytic performance. In combination with its ease for multiplexing, it also has applications as a convenient platform for assessing patient fibrinolytic potential and screening thrombolytic drug activities in personalized medical applications.

In-vitro thromboelastographic characterization of reconstituted whole blood utilizing cryopreserved platelets.

Conducting in-vitro thrombosis research presents numerous challenges, the primary of which is working with blood products, whether whole blood or fractionated whole blood, that have limited functional shelf-lives. As a result, being able to significantly prolong the clotting functionality of whole blood via fractionation and recombination promises greater accessibility via resource minimization in the realm of thrombosis research. Whole blood with CPDA1 from healthy volunteers was fractionated and stored as frozen platelet-free plasma (PFP, -20°C), refrigerated packed red blood cells (pRBCs, 4°C) and cryopreserved platelets (-80°C). Subsequent recombination of the above components into their native ratios were tested via thromboelastography (TEG) to capture clotting dynamics over a storage period of 13 weeks in comparison to refrigerated unfractionated WB+CPDA1. Reconstituted whole blood utilizing PFP, pRCBs and cryopreserved platelets were able to maintain clot strength (maximum amplitude) akin to day-0 whole blood even after 13 weeks of storage. Clots formed by reconstituted whole blood exhibited quicker clotting dynamics with nearly two-fold shorter R-times and nearly 1.3-fold increase in fibrin deposition rate as measured by TEG. Storage of fractionated whole blood components, in their respective ideal conditions, provides a means of prolonging the usable life of whole blood for in-vitro thrombosis research. Cryopreserved platelets, when recombined with frozen PFP and refrigerated pRBCs, are able to form clots that nearly mirror the overall clotting profile expected of freshly drawn WB.

Environmental microplastic and nanoplastic: Exposure routes and effects on coagulation and the cardiovascular system.

Plastic pollution has been a growing concern in recent decades due to the proliferation and ease of manufacturing of single use plastic products and inadequate waste and recycling management. Microplastic, and even smaller nanoplastic, particles are persistent pollutants in aquatic and terrestrial systems and are the subject of active and urgent research. This review will explore the current research on how exposure to plastic particles occurs and the risks associated from different exposure routes: ingestion, inhalation, and dermal exposure. The effects of microplastics on the cardiovascular system are of particular importance due to its sensitivity and ability to transport particles to other organ systems. The effects of microplastics and nanoplastics on the heart, platelet aggregation, and thrombus formation will all be explored with focus on how the particle characteristics modulate their effect. Plastic particle interactions are highly dependent on both their size and their surface chemistry and interesting research is being done with the interaction of particle characteristics and effect on thrombosis and the cardiovascular system. There is significant uncertainty surrounding some of the findings in this field as research in this area is still maturing. There are undoubtedly more physiological consequences than we are currently aware of resulting from environmental plastic exposure and more studies need to be conducted to reveal the full extent of pathologies caused by the various routes of microplastic exposure, with particular emphasis on longitudinal exposure effects. Further research will allow us to recognize the full extent of physiological impact and begin developing viable solutions to reduce plastic pollution and potentially design interventions to mitigate in-vivo plastic effects following significant or prolonged exposure.

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization.

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients' hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

Inhaled nitric oxide to control platelet hyper-reactivity in patients with acute submassive pulmonary embolism.

BACKGROUND: We test if inhaled nitric oxide (NO) attenuates platelet functional and metabolic hyper-reactivity in subjects with submassive pulmonary embolism (PE). METHODS: Participants with PE were randomized to either 50 ppm NO + O2 or O2 only for 24 h with blood sampling at enrollment and after treatment; results were compared with healthy controls. Platelet metabolic activity was assessed by oxygen consumption (basal and uncoupled) and reactivity was assessed with agonist-stimulated thromboelastography (TEG) and fluorometric measurement of agonist-stimulated cytosolic [Ca++] without and with pharmacological soluble guanylate (sGC) modulation. RESULTS: Participants (N = 38 per group) were well-matched at enrollment for PE severity, comorbidities as well as TEG parameters and platelet O2 consumption. NO treatment doubled the mean plasma [NO3-] (P < 0.001) indicating successful delivery, but placebo treatment produced no change. After 24 h, neither TEG nor O2 consumption parameters differed significantly between treatment groups. Platelet cytosolic [Ca++] was elevated with PE versus controls, and was decreased by treatment with cinaciguat (an sGC activator), but not riociguat (an sGC stimulator). Stimulated platelet lysate sGC activity was increased with PE compared with controls. CONCLUSIONS: In patients with acute submassive PE, despite evidence of adequate drug delivery, inhaled NO had no major effect on platelet O2 consumption or agonist-stimulated parameters on TEG. Pharmacological activation, but not stimulation, of sGC effectively decreased platelet cytosolic [Ca++], and platelet sGC activity was increased with PE, confirming the viability of sGC as a therapeutic target.

Fibrin clot formation under diverse clotting conditions: Comparing turbidimetry and thromboelastography.

Thrombosis is a leading cause of death around the world. Fibrin, the protein primarily responsible for clot formation, is formed via cleaving soluble fibrinogen by thrombin with resulting properties varying under different clot forming conditions. This study sought to compare trends across thromboelastography (TEG) and turbidimetry utilizing a simplified fibrinogen/thrombin clot model. Turbidimetry is an optical measure (550 nm) of fibrin clot formation and is widely utilized due to its laboratory accessibility and ease of use. Thromboelastography (TEG) is a specialized viscoelastic technique that directly measures clot strength and is primarily utilized in the clinical setting to assess patients' hemostasis. In these experiments, human and bovine fibrin clots were formed in-vitro by mixing fibrinogen and thrombin under diverse clotting conditions. Increasing thrombin concentration (0 to 10 U/mL), ionic strength (0.05 to 0.3 M), pH (5.5 to 8.1), and lowering albumin concentration (100 to 0 mg/mL) resulted in decreased clot turbidity and increased clot strength using species-matched bovine and human fibrinogen and thrombin. Whereas, increasing fibrinogen concentration (1 to 5 mg/mL) resulted in increased clot turbidity and increased clot strength for both species-matched and cross-species fibrinogen and thrombin. Clotting times with both techniques followed a similar trend and were observed to be unchanged when varying albumin concentration, elongated with increasing fibrinogen, and shortened with increasing pH, ionic strength, and thrombin. TEG and turbidimetry track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

Antibody conjugation and formulation.

In an era where ultra-high antibody concentrations, high viscosities, low volumes, auto-injectors and long storage requirements are already complex problems with the current unconjugated monoclonal antibodies on the market, the formulation demands for antibody-drug conjugates (ADCs) are significant. Antibodies have historically been administered at relatively low concentrations through intravenous (IV) infusion due to their large size and the inability to formulate for oral delivery. Due to the high demands associated with IV infusion and the development of novel antibody targets and unique antibody conjugates, more accessible routes of administration such as intramuscular and subcutaneous are being explored. This review will summarize various site-specific and non-site-specific antibody conjugation techniques in the context of ADCs and the demands of formulation for high concentration clinical implementation.

Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase.

While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.

Disinhibiting neurons in the dorsomedial hypothalamus delays the onset of exertional fatigue and exhaustion in rats exercising in a warm environment.

Stimulants cause hyperthermia, in part, by increasing heat generation through exercise. Stimulants also delay the onset of fatigue and exhaustion allowing animals to exercise longer. If used in a warm environment, this combination (increased exercise and decreased fatigue) can cause heat stroke. The dorsomedial hypothalamus (DMH) is involved in mediating locomotion from stimulants. Furthermore, inhibiting the DMH decreases locomotion and prevents hyperthermia in rats given stimulants in a warm environment. Whether the DMH is involved in mediating exercise-induced fatigue and exhaustion is not known. We hypothesized that disinhibiting neurons in the dorsomedial hypothalamus (DMH) would delay the onset of fatigue and exhaustion in animals exercising in a warm environment. To test this hypothesis, we used automated video tracking software to measure fatigue and exhaustion. In rats, using wearable mini-pumps, we demonstrated that disinhibiting the DMH, via bicuculline perfusion (5 µM), increased the duration of exercise in a warm environment as compared to control animals (25 ±â€¯3 min vs 15 ±â€¯2 min). Bicuculline-perfused animals also had higher temperatures at exhaustion (41.4 ±â€¯0.2 °C vs 40.0 ±â€¯0.4 °C). Disinhibiting neurons in the DMH also increased the time to fatigue. Our data show that the same region of the hypothalamus that is involved in mediating locomotion to stimulants, is also involved in controlling exhaustion and fatigue. These findings have implications for understanding the cause and treatment of stimulant-induced-hyperthermia.

Printed Graphene Electrochemical Biosensors Fabricated by Inkjet Maskless Lithography for Rapid and Sensitive Detection of Organophosphates.

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/µM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms.