N-Cadherin Targeted Melanin Nanoparticles Reverse the Endothelial-Mesenchymal Transition in Vascular Endothelial Cells to Potentially Slow the Progression of Atherosclerosis and Cancer.

Endothelial-mesenchymal transition (EndoMT) of vascular endothelial cells has recently been considered as a key player in the early progression of a variety of vascular and nonvascular diseases, including atherosclerosis, cancer, and organ fibrosis. However, current strategies attempting to identify pharmacological inhibitors to block the regulatory pathways of EndoMT suffer from poor selectivity, unwanted side effects, and a heterogeneous response from endothelial cells with different origins. Furthermore, EndoMT inhibitors focus on preventing EndoMT, leaving the endothelial cells that have already undergone EndoMT unresolved. Here, we report the design of a simple but powerful nanoparticle system (i.e., N-cadherin targeted melanin nanoparticles) to convert cytokine-activated, mesenchymal-like endothelial cells back to their original endothelial phenotype. We term this process "Reversed EndoMT" (R-EndoMT). R-EndoMT allows the impaired endothelial barriers to recover their quiescence and intactness, with significantly reduced leukocyte and cancer cell adhesion and transmigration, which could potentially stop atheromatous plaque formation and cancer metastasis in the early stages. R-EndoMT is achieved on different endothelial cell types originating from arteries, veins, and capillaries, independent of activating cytokines. We reveal that N-cadherin targeted melanin nanoparticles reverse EndoMT by downregulating an N-cadherin dependent RhoA activation pathway. Overall, this approach offers a different prospect to treat multiple EndoMT-associated diseases by designing nanoparticles to reverse the phenotypical transition of endothelial cells.

Plasmonic biochips with enhanced stability in harsh environments for the sensitive detection of prostate-specific antigen.

Hollow and porous plasmonic nanomaterials have been demonstrated for highly sensitive biosensing applications due to their distinctive optical properties. Immunosensors, which rely on antibody-antigen interactions, are essential constituents of diverse biosensing platforms owing to their exceptional binding affinity and selectivity. The majority of immunosensors and conventional bioassays needs special storage conditions and cold chain systems for transportation. Prostate-specific antigen (PSA), a serine protease, is widely employed in the diagnosis of prostate cancer. In this study, we present the successful utilization of a biopolymer-preserved plasmonic biosensor with improved environmental stability for the sensitive detection of PSA. The preserved plasmonic biosensors exhibited sustained sensitivity in the detection of PSA, achieving a limit of detection of 10 pg mL-1. Furthermore, these biosensors exhibited remarkable stability at elevated temperatures for one week.

Low-dose albumin-coated gold nanorods induce intercellular gaps on vascular endothelium by causing the contraction of cytoskeletal actin.

Cytotoxicity of nanoparticles, typically evaluated by biochemical-based assays, often overlook the cellular biophysical properties such as cell morphology and cytoskeletal actin, which could serve as more sensitive indicators for cytotoxicity. Here, we demonstrate that low-dose albumin-coated gold nanorods (HSA@AuNRs), although being considered noncytotoxic in multiple biochemical assays, can induce intercellular gaps and enhance the paracellular permeability between human aortic endothelial cells (HAECs). The formation of intercellular gaps can be attributed to the changed cell morphology and cytoskeletal actin structures, as validated at the monolayer and single cell levels using fluorescence staining, atomic force microscopy, and super-resolution imaging. Molecular mechanistic study shows the caveolae-mediated endocytosis of HSA@AuNRs induces the calcium influx and activates actomyosin contraction in HAECs. Considering the important roles of endothelial integrity/dysfunction in various physiological/pathological conditions, this work suggests a potential adverse effect of albumin-coated gold nanorods on the cardiovascular system. On the other hand, this work also offers a feasible way to modulate the endothelial permeability, thus promoting drug and nanoparticle delivery across the endothelium.

Photothermal Attenuation of Cancer Cell Stemness, Chemoresistance, and Migration Using CD44-Targeted MoS2 Nanosheets.

Cancer stem-like cells (CSCs) play key roles in chemoresistance, tumor metastasis, and clinical relapse. However, current CSC inhibitors lack specificity, efficacy, and applicability to different cancers. Herein, we introduce a nanomaterial-based approach to photothermally induce the differentiation of CSCs, termed "photothermal differentiation", leading to the attenuation of cancer cell stemness, chemoresistance, and metastasis. MoS2 nanosheets and a moderate photothermal treatment were applied to target a CSC surface receptor (i.e., CD44) and modulate its downstream signaling pathway. This treatment forces the more stem-like cancer cells to lose the mesenchymal phenotype and adopt an epithelial, less stem-like state, which shows attenuated self-renewal capacity, more response to anticancer drugs, and less invasiveness. This approach could be applicable to various cancers due to the broad availability of the CD44 biomarker. The concept of using photothermal nanomaterials to regulate specific cellular activities driving the differentiation of CSCs offers a new avenue for treating refractory cancers.

"Non-cytotoxic" doses of metal-organic framework nanoparticles increase endothelial permeability by inducing actin reorganization.

Cytotoxicity of nanoparticles is routinely characterized by biochemical assays such as cell viability and membrane integrity assays. However, these approaches overlook cellular biophysical properties including changes in the actin cytoskeleton, cell stiffness, and cell morphology, particularly when cells are exposed to "non-cytotoxic" doses of nanoparticles. Zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NPs), a member of metal-organic framework family, has received increasing interest in various fields such as environmental and biomedical sciences. ZIF-8 NPs may enter the blood circulation system after unintended oral and inhalational exposure or intended intravenous injection for diagnostic and therapeutic applications, yet the effect of ZIF-8 NPs on vascular endothelial cells is not well understood. Here, the biophysical impact of "non-cytotoxic" dose ZIF-8 NPs on human aortic endothelial cells (HAECs) is investigated. We demonstrate that "non-cytotoxic" doses of ZIF-8 NPs, pre-defined by a series of biochemical assays, can increase the endothelial permeability of HAEC monolayers by causing cell junction disruption and intercellular gap formation, which can be attributed to actin reorganization within adjacent HAECs. Nanomechanical atomic force microscopy and super resolution fluorescence microscopy further confirm that "non-cytotoxic" doses of ZIF-8 NPs change the actin structure and cell morphology of HAECs at the single cell level. Finally, the underlying mechanism of actin reorganization induced by the "non-cytotoxic" dose ZIF-8 NPs is elucidated. Together, this study indicates that the "non-cytotoxic" doses of ZIF-8 NPs, intentionally or unintentionally introduced into blood circulation, may still pose a threat to human health, considering increased endothelial permeability is essential to the progression of a variety of diseases. From a broad view of cytotoxicity evaluation, it is important to consider the biophysical properties of cells, since they can serve as novel and more sensitive markers to assess nanomaterial's cytotoxicity.

Spectral characterization of cell surface motion for mechanistic investigations of cellular mechanobiology.

Understanding the specific mechanisms responsible for anabolic and catabolic responses to static or dynamic force are largely poorly understood. Because of this, most research groups studying mechanotransduction due to dynamic forces employ an empirical approach in deciding what frequencies to apply during experiments. While this has been shown to elucidate valuable information regarding how cells respond under controlled provocation, it is often difficult or impossible to determine a true optimal frequency for force application, as many intracellular complexes are involved in receiving, propagating, and responding to a given stimulus. Here we present a novel adaptation of an analytical technique from the fields of civil and mechanical engineering that may open the door to direct measurement of mechanobiological cellular frequencies which could be used to target specific cell signaling pathways leveraging synergy between outside-in and inside-out mechanotransduction approaches. This information could be useful in identifying how specific proteins are involved in the homeostatic balance, or disruption thereof, of cells and tissue, furthering the understanding of the pathogenesis and progression of many diseases across a wide variety of cell types, which may one day lead to the development of novel mechanobiological therapies for clinical use.

Reversing the Epithelial-Mesenchymal Transition in Metastatic Cancer Cells Using CD146-Targeted Black Phosphorus Nanosheets and a Mild Photothermal Treatment.

Cancer metastasis leads to most deaths in cancer patients, and the epithelial-mesenchymal transition (EMT) is the key mechanism that endows the cancer cells with strong migratory and invasive abilities. Here, we present a nanomaterial-based approach to reverse the EMT in cancer cells by targeting an EMT inducer, CD146, using engineered black phosphorus nanosheets (BPNSs) and a mild photothermal treatment. We demonstrate this approach can convert highly metastatic, mesenchymal-type breast cancer cells to an epithelial phenotype (i.e., reversing EMT), leading to a complete stoppage of cancer cell migration. By using advanced nanomechanical and super-resolution imaging, complemented by immunoblotting, we validate the phenotypic switch in the cancer cells, as evidenced by the altered actin organization and cell morphology, downregulation of mesenchymal protein markers, and upregulation of epithelial protein markers. We also elucidate the molecular mechanism behind the reversal of EMT. Our results reveal that CD146-targeted BPNSs and a mild photothermal treatment synergistically contribute to EMT reversal by downregulating membrane CD146 and perturbing its downstream EMT-related signaling pathways. Considering CD146 overexpression has been confirmed on the surface of a variety of metastatic, mesenchymal-like cancer cells, this approach could be applicable for treating various cancer metastasis via modulating the phenotype switch in cancer cells.

Transmembrane MUC18 Targeted Polydopamine Nanoparticles and a Mild Photothermal Effect Synergistically Disrupt Actin Cytoskeleton and Migration of Cancer Cells.

Transmembrane MUC18 is highly expressed on most metastatic cancers. Herein, we demonstrate that targeting MUC18 with polydopamine nanoparticles (PDA NPs) and a mild photothermal effect can completely cease the migration of melanoma and breast cancer cells without killing the cells. The inhibited cell migration can be attributed to the altered actin cytoskeleton, cell stiffness, and cell morphology, as revealed by nanomechanical and super resolution fluorescence imaging techniques. Further mechanistic studies at the molecular level show that MUC18 targeted PDA NPs and a mild photothermal treatment produce a synergistic effect on the actin cytoskeleton by downregulating the transmembrane MUC18 and interrupting ezrin-radixin-moesin phosphorylation, thereby releasing the actin cytoskeleton from the cell membrane and compromising force transduction through the actin cytoskeleton to the transmembrane MUC18. Overall, the concept of targeting transmembrane metastatic markers and disrupting their downstream effectors (i.e., actin and actin-binding proteins) opens up a new avenue to cancer therapy.

Targeting cancer cell adhesion molecule, CD146, with low-dose gold nanorods and mild hyperthermia disrupts actin cytoskeleton and cancer cell migration.

Cluster of differentiation 146 (CD146), a cancer cell adhesion molecule, is over-expressed on the surfaces of melanoma, breast, ovarian, and prostate cancer cells, and its high expression indicates the migration tendency of these cancer cells and poor patient prognosis. Here, we hypothesize that targeting the CD146 with low-dose gold nanorods combined with mild hyperthermia can stop the migration of these cancer cells. Two metastatic cancer cells including a melanoma and a breast cancer cell line are selected as the model systems. Cell migration assays show that the migration of both cell lines can be completely stopped by the treatment. Atomic force microscopy and super resolution fluorescence microscopy reveal the alterations of actin cytoskeleton and cell morphology correspond to the inhibited cell migration. Further mechanistic analysis indicates the treatment disrupts the actin cytoskeleton by a synergistic mechanism including depleting membrane CD146 and interfering ezrin-radixin-moesin phosphorylation. As a result, we believe targeting CD146 with low-dose gold nanorods and mild hyperthermia could be a versatile, effective, and safe approach for stopping cancer metastasis. More broadly, the concept of targeting cancer cell surface markers that connect the underlying actin cytoskeleton, offers enormous potential in treating cancer metastasis, which accounts for more than 90% of cancer-associated mortality.

Low doses of zeolitic imidazolate framework-8 nanoparticles alter the actin organization and contractility of vascular smooth muscle cells.

Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles have emerged as a promising platform for drug delivery and controlled release. Considering most ZIF-8 nanoparticle drug carriers are designed to be administered intravenously, and thus would directly contact vascular smooth muscle cells (VSMCs) in many circumstances, the potential interactions of ZIF-8 nanoparticles with VSMCs require investigation. Here, the effects of low doses of ZIF-8 nanoparticles on VSMC morphology, actin organization, and contractility are investigated. Two nanoscale imaging tools, atomic force microscopy, and direct stochastic optical reconstruction microscopy, show that even at the concentrations (12.5 and 25 µg/ml) that were deemed "safe" by conventional biochemical cell assays (MTT and LDH assays), ZIF-8 nanoparticles can still cause changes in cell morphology and actin cytoskeleton organization at the cell apical and basal surfaces. These cytoskeletal structural changes impair the contractility function of VSMCs in response to Angiotensin II, a classic vasoconstrictor. Based on intracellular zinc and actin polymerization assays, we conclude that the increased intracellular Zn2+ concentration due to the uptake and dissociation of ZIF-8 nanoparticles could cause the actin cytoskeleton dis-organization, as the elevated Zn2+ directly disrupts the actin assembly process, leading to altered actin organization such as branches and networks. Since the VSMC phenotype change and loss of contractility are fundamental to the development of atherosclerosis and related cardiovascular diseases, it is worth noting that these low doses of ZIF-8 nanoparticles administered intravenously could still be a safety concern in terms of cardiovascular risks. Moving forward, it is imperative to re-consider the "safe" nanoparticle dosages determined by biochemical cell assays alone, and take into account the impact of these nanoparticles on the biophysical characteristics of VSMCs, including changes in the actin cytoskeleton and cell morphology.

Stabilization of surface-bound antibodies for ELISA based on a reversable zeolitic imidazolate framework-8 coating.

Immunoassays typically must be stored under refrigerated conditions because antibodies, after being immobilized to solid surfaces, tend to lose their recognition capabilities to target antigens under non-refrigerated conditions. This requirement hinders application of immunoassays in resource-limited settings including rural clinics in tropical regions, disaster struck areas, and low-income countries, where refrigeration may not be feasible. In this work, a facile approach based on a reversable zeolitic imidazolate framework-8 (ZIF-8) coating is introduced to stabilize surface-bound antibodies on enzyme-linked immunosorbent assay (ELISA) plates under non-refrigerated conditions. Using a sandwich ELISA for the detection of neutrophil gelatinase-associated lipocalin (NGAL), a urine biomarker for acute kidney injury, as a model system, ZIF-8 is demonstrated to be able to uniformly coat the surface-bound anti-NGAL IgG, and stabilize the dynamic range and detection sensitivity of the assay after storage at an elevated temperature (50 °C) for at least 4 weeks. The stabilization efficacy of the ZIF-8 coating is comparable to the current "gold standard" refrigeration approach, and superior to the commonly used sucrose coating method. This approach will greatly improve the shelf-life and stability of antibody-coated ELISAs and other types of assays which utilize surface-bound antibodies, thus extending biomedical research and medical diagnostics to resource-limited settings.

Metal-Organic Framework Preserves the Biorecognition of Antibodies on Nanoscale Surfaces Validated by Single-Molecule Force Spectroscopy.

Antibody biorecognition forms the basis for numerous biomedical applications such as diagnostic assays, targeted drug delivery, and targeted cancer imaging. However, antibodies, especially after being conjugated to surfaces or nanostructures, suffer from stability issues when stored under nonrefrigeration conditions. Therefore, enhancing the stability of antibodies on surfaces and nanostructures under ambient and elevated temperatures is of paramount importance for many nanobiotechnology applications. In this study, we introduce a simple and facile approach based on a metal-organic framework (MOF) coating to preserve the biorecognition capability of antibodies immobilized on nanoscale surfaces after exposure to elevated temperatures for a prolonged period. By using atomic force microscopy (AFM)-based force spectroscopy, we demonstrate that the MOF coating is able to preserve the binding force and binding frequency of the anti-CD-146 antibody attached to an AFM tip to CD-146 antigen on the surface of melanoma cells at the single-molecule level. We also demonstrate that the MOF coating outperforms another commonly used sucrose coatings in terms of maintaining the binding force and binding frequency of the antibody to antigen. Herein, the AFM tip functionalized with antibodies provides a nanoscale testbed (analogous to an antibody-conjugated nanostructure) to assess antibody biorecognition at the single-molecule level and preservation efficacy under antibody denaturing conditions. This MOF coating approach should be applicable to the preservation of a variety of antibody-conjugated nanostructures aiming for targeted drug delivery, targeted cancer imaging, and nanobiosensors. The improved stability and elimination of refrigeration requirements will facilitate wide applications of antibody-enabled nanobiotechnology in resource-limited environments and populations.

Bioplasmonic paper-based assay for perilipin-2 non-invasively detects renal cancer.

Renal cell carcinoma (RCC) has poor survival prognosis because it is asymptomatic at an early, more curative stage. Recently, urine perilipin-2 (PLIN-2) was demonstrated to be a sensitive and specific biomarker for the noninvasive, early detection of RCC and an indispensable indicator to distinguish cancer from a benign renal mass. However, current Western blot or ELISA PLIN-2 assays are complicated, expensive, time-consuming or insensitive, making them unsuitable for routine analysis in clinical settings. Here we developed a plasmonic biosensor based on the high refractive index sensitivity of gold nanorattles for the rapid detection of PLIN-2 in patient urine. The paper-based plasmonic assay is highly sensitive and has a dynamic range of 50 pg/ml to 5 µg/ml PLIN-2. The assay is not compromised by variations in urine pH or high concentrations of interfering proteins such as albumin and hemoglobin, making it an excellent candidate for routine clinical applications. The urine PLIN-2 assay readily distinguished patients with pathologically proven clear cell carcinomas of various size, stage and grade (55.9 [39.5, 75.8] ng/ml, median [1st and 3rd quartile]) from age-matched controls (0.3 [0.3, 0.5] ng/ml), patients with bladder cancer (0.5 [0.4, 0.6] ng/ml) and patients with diabetic nephropathy (0.6 [0.4, 0.7] ng/ml). Urine PLIN-2 concentrations were roughly proportional to tumor size (Pearson coefficient 0.59). Thus, this cost-effective and label-free method represents a novel approach to conduct a non-invasive population screen or rapid differential diagnosis of imaged renal masses, significantly facilitating the early detection and diagnosis of RCC.

Metal-Organic Framework Encapsulation Preserves the Bioactivity of Protein Therapeutics.

Protein therapeutics are prone to lose their structure and bioactivity under various environmental stressors. This study reports a facile approach using a nanoporous material, zeolitic imidazolate framework-8 (ZIF-8), as an encapsulant for preserving the prototypic protein therapeutic, insulin, against different harsh conditions that may be encountered during storage, formulation, and transport, including elevated temperatures, mechanical agitation, and organic solvent. Both immunoassay and spectroscopy analyses demonstrate the preserved chemical stability and structural integrity of insulin offered by the ZIF-8 encapsulation. Biological activity of ZIF-8-preserved insulin after storage under accelerated degradation conditions (i.e., 40 °C) is evaluated in vivo using a diabetic mouse model, and shows comparable bioactivity to refrigeration-stored insulin (-20 °C). It is also demonstrated that ZIF-8-preserved insulin has low cytotoxicity in vitro and does not cause side effects in vivo. Furthermore, ZIF-8 residue can be completely removed by a simple purification step before insulin administration. This biopreservation approach is potentially applicable to diverse protein therapeutics, thus extending the benefits of advanced biologics to resource-limited settings and underserved populations/regions.

Ultrarobust Biochips with Metal-Organic Framework Coating for Point-of-Care Diagnosis.

Most biosensors relying on antibodies as recognition elements fail in harsh environment conditions such as elevated temperatures, organic solvents, or proteases because of antibody denaturation, and require strict storage conditions with defined shelf life, thus limiting their applications in point-of-care and resource-limited settings. Here, a metal-organic framework (MOF) encapsulation is utilized to preserve the biofunctionality of antibodies conjugated to nanotransducers. This study investigates several parameters of MOF coating (including growth time, surface morphology, thickness, and precursor concentrations) that determine the preservation efficacy against different protein denaturing conditions in both dry and wet environments. A plasmonic biosensor based on gold nanorods as the nanotransducers is employed as a model biodiagnostic platform. The preservation efficacy attained through MOF encapsulation is compared to two other commonly employed materials (sucrose and silk fibroin). The results show that MOF coating outperforms sucrose and silk fibroin coatings under several harsh conditions including high temperature (80 °C), dimethylformamide, and protease solution, owing to complete encapsulation, stability in wet environment and ease of removal at point-of-use by the MOF. We believe this study will broaden the applicability of this universal approach for preserving different types of on-chip biodiagnostic reagents and biosensors/bioassays, thus extending the benefits of advanced diagnostic technologies in resource-limited settings.

Add-on plasmonic patch as a universal fluorescence enhancer.

Fluorescence-based techniques are the cornerstone of modern biomedical optics, with applications ranging from bioimaging at various scales (organelle to organism) to detection and quantification of a wide variety of biological species of interest. However, the weakness of the fluorescence signal remains a persistent challenge in meeting the ever-increasing demand to image, detect, and quantify biological species with low abundance. Here, we report a simple and universal method based on a flexible and conformal elastomeric film with adsorbed plasmonic nanostructures, which we term a "plasmonic patch," that provides large (up to 100-fold) and uniform fluorescence enhancement on a variety of surfaces through simple transfer of the plasmonic patch to the surface. We demonstrate the applications of the plasmonic patch in improving the sensitivity and limit of detection (by more than 100 times) of fluorescence-based immunoassays implemented in microtiter plates and in microarray format. The novel fluorescence enhancement approach presented here represents a disease, biomarker, and application agnostic ubiquitously applicable fundamental and enabling technology to immediately improve the sensitivity of existing analytical methodologies in an easy-to-handle and cost-effective manner, without changing the original procedures of the existing techniques.

Nanoscale characterization of forensically relevant epithelial cells and surface associated extracellular DNA.

Atomic force microscopy provides a novel morphological and physico-chemical perspective to analyze epithelial cell samples in forensic investigations. As a nanoscale, single cell tool, it allows the investigation of scarce samples in a non-destructive fashion. Using chemical force spectroscopy, it permits the identification of specific functional groups or surface molecules. Of specific interest is the presence of extracellular DNA (eDNA) on the surface of epithelial cells that line the exterior skin and interior cavities of human bodies, and can transfer onto surfaces through contact with skin and saliva. To date, this eDNA has only been measured a bulk level. Here, using nanoscale imaging, we first describe the unique differences between keratinized epithelial cells and non-keratinized buccal cells. Then via a force mapping technique, we show how eDNA can be spatially located and quantified on the cell surface. Our results suggest that presence and relative quantity of surface-associated, extracellular DNA signatures can be analyzed on individual epithelial cells from different tissue sources, providing a new tool in the forensic analysis of touch samples.

Single Molecule Force Spectroscopy to Compare Natural versus Artificial Antibody-Antigen Interaction.

Biorecognition is central to various biological processes and finds numerous applications in virtually all areas of chemistry, biology, and medicine. Artificial antibodies, produced by imprinting synthetic polymers, are designed to mimic the biological recognition capability of natural antibodies, while exhibiting superior thermal, chemical, and environmental stability compared to their natural counterparts. The binding affinity of the artificial antibodies to their antigens characterizes the biorecognition ability of these synthetic nanoconstructs and their ability to replace natural recognition elements. However, a quantitative study of the binding affinity of an artificial antibody to an antigen, especially at the molecular level, is still lacking. In this study, using atomic force microscopy-based force spectroscopy, the authors show that the binding affinity of an artificial antibody to an antigen (hemoglobin) is weaker than that of natural antibody. The fine difference in the molecular interactions manifests into a significant difference in the bioanalytical parameters of biosensors based on these recognition elements.

Metal-Organic Framework as a Protective Coating for Biodiagnostic Chips.

Zeolitic imidazolate framework-8 (ZIF-8) grown around antibodies anchored to plasmonic nanostructures serves as a protective layer to preserve the biorecognition ability of antibodies stored at room and elevated temperatures for several days. The biofunctionality of the ZIF-8-protected biochip can be restored by a simple water-rinsing step, making it highly convenient for use in point-of-care and resource-limited settings.

Silk-Encapsulated Plasmonic Biochips with Enhanced Thermal Stability.

Because of their high sensitivity, cost-efficiency, and great potential as point-of-care biodiagnostic devices, plasmonic biosensors based on localized surface plasmon resonance have gained immense attention. However, most plasmonic biosensors and conventional bioassays rely on natural antibodies, which are susceptible to elevated temperatures and nonaqueous media. Hence, an expensive and cumbersome "cold chain" system is necessary to preserve the labile antibodies by maintaining optimal cold temperatures during transport, storage, and handling. Herein, we introduce a facile approach to preserve the antibody activity on a biosensor surface even at elevated temperatures. We show that silk fibroin film could be used as a protective layer to preserve the activity of a model antibody (Rabbit IgG) and cardiac troponin antibody at both room temperature and 40 °C over several days. Furthermore, a simple aqueous rinsing process restores the biofunctionality of the biosensor. This energy-efficient and environmentally friendly method represents a novel approach to eliminate the cold chain and temperature-controlled packing of diagnostic reagents and materials, thereby extending the capability of antibody-based biosensors to different resource-limited circumstances such as developing countries, an ambulance, an intensive care unit emergency room, and battlefield.