Mitochondrial Dynamics in Neurodegenerative Diseases: Unraveling the Role of Fusion and Fission Processes.

Neurodegenerative diseases (NDs) are a diverse group of disorders characterized by the progressive degeneration and death of neurons, leading to a range of neurological symptoms. Despite the heterogeneity of these conditions, a common denominator is the implication of mitochondrial dysfunction in their pathogenesis. Mitochondria play a crucial role in creating biomolecules, providing energy through adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS), and producing reactive oxygen species (ROS). When they're not functioning correctly, becoming fragmented and losing their membrane potential, they contribute to these diseases. In this review, we explore how mitochondria fuse and undergo fission, especially in the context of NDs. We discuss the genetic and protein mutations linked to these diseases and how they impact mitochondrial dynamics. We also look at the key regulatory proteins in fusion (MFN1, MFN2, and OPA1) and fission (DRP1 and FIS1), including their post-translational modifications. Furthermore, we highlight potential drugs that can influence mitochondrial dynamics. By unpacking these complex processes, we aim to direct research towards treatments that can improve life quality for people with these challenging conditions.

Current progress, strategy, and prospects of PD-1/PDL-1 immune checkpoint biosensing platforms for cancer diagnostics, therapy monitoring, and drug screening.

Recent technological advancements in testing and monitoring instrumentation have greatly contributed to the progress in cancer treatment by surgical, chemotherapeutic and radiotherapeutic interventions. However, the mortality rate still remains high, calling for the development of new treatment strategies with higher efficacy. Extensive efforts driven in this direction have included broadening of early cancer screening and applying innovative theranostic nanotechnologies. They have been supported by platforms introduced to enable the detection and monitoring of cancer biomarkers, inhibitors, and other agents, able to slow down cancer progression and prevent metastasis. Despite of the well-recognized principles of the immune checkpoint blockade, the efficacy of immunotherapy achieved so far does not meet the well-founded expectations. For a successful cancer treatment, highly sensitive, robust, and inexpensive multiplex biosensors have to be designed to aid in the biomarkers monitoring and in the development of new inhibitors. In this review, we provide an overview of the efforts undertaken to aid in the development and monitoring of anticancer immunotherapy, based on the programmed cell-death immune checkpoint (PD-1/PDL-1) blockade, by designing biosensors for the detection of relevant cancer biomarkers and their inhibitors screening. This review also emphasizes alternative targets made by exosomes carrying PD-L1 overexpressed in cancer cells and passed into the excreted exosomes. Evaluated are also novel targeted drug delivery nanocarriers, providing simultaneous biosensing, thereby contributing to the emerging immune checkpoint cancer therapy. On the basis of the current trends and the emerging technologies, future perspectives of cancer diagnostics and treatment monitoring using biosensing platforms are projected.

DNA Aptamer Beacon Probe (ABP) for Monitoring of Adenosine Triphosphate Level in SW480 Cancer Cells Treated with Glycolysis Inhibitor 2-Deoxyglucose.

Early cancer screening enables timely detection of carcinogenesis, and aids in prompt clinical intervention. Herein, we report on the development of a simple, sensitive, and rapid fluorometric assay based on the aptamer probe (aptamer beacon probe, ABP) for monitoring the energy-demand biomarker adenosine triphosphate (ATP), an essential energy source that is released into the tumor microenvironment. Its level plays a significant role in risk assessment of malignancies. The operation of the ABP for ATP was examined using solutions of ATP and other nucleotides (UTP, GTP, CTP), followed by monitoring of ATP production in SW480 cancer cells. Then, the effect of a glycolysis inhibitor, 2-deoxyglucose (2-DG), on SW480 cells was investigated. The stability of predominant ABP conformations in the temperature range of 23-91 °C and the effects of temperature on ABP interactions with ATP, UTP, GTP, and CTP were evaluated based on quenching efficiencies (QE) and Stern-Volmer constants (KSV). The optimized temperature for best selectivity of ABP toward ATP was 40 °C (KSV = 1093 M-1, QE = 42%). We have found that the inhibition of glycolysis in SW480 cancer cells by 2-deoxyglucose resulted in lowering of ATP production by 31.7%. Therefore, monitoring and modulation of ATP concentration may aid in future cancer treatment.

Effects of Supplementation with an Herbal Mixture on the Antioxidant Capacity of Milk.

The aim of this study was to assess the effect of the addition of a standardized herbal mixture to the feed ration for Holstein-Friesian cows on the antioxidant capacity of milk. The study was carried out on a farm specialized in breeding dairy cattle. The exact study involved 30 cows in lactation III, which were in the first phase of lactation at the beginning of the experiment (15 cows-control group; 15 cows-experimental group). The nutrition supplied to the cows was based on the TMR (total mixed ration) system, with roughage and concentrate fodder used as the basis of the feed ration. The addition of a standardized blend of dried herbs, i.e., oregano (Origanum vulgare), thyme (Thymus vulgaris), purple coneflower (Echinacea purpurea), and cinnamon bark (Cinnamomum zeylanicum), was the experimental factor. Powdered herbs were administered as a component of the concentrate fodder at the dose of 3% DM ration/day/head. Milk samples were collected four times during the experiment (term 0 after the colostrum period and then after lactation weeks 2, 4, and 6). The following parameters were determined in the milk: the basic chemical composition, i.e., the content of total protein, fat, lactose, and casein; somatic cell count; content of selected whey proteins (α-lactalbumin, ß-lactoglobulin, lactoferrin, BSA); and fat-soluble vitamins (A, D3, E). Additionally, the milk antioxidant capacity (ABTS, FRAP, DPPH) was determined and the degree of antioxidant protection (DAP) was calculated. It was shown that the milk from cows receiving the herbal blend-supplemented fodder had a higher content of casein, compared to the control group. The herbal supplementation contributed to a significant increase in the content of bioactive compounds, i.e., selected whey proteins (ß-lactoglobulin, lactoferrin) and lipophilic vitamins (A, E). The milk was also characterized by significantly higher antioxidant potential (regardless of the measurement method) and a higher degree of antioxidant protection (DAP).

Effective Optical Image Assessment of Cellulose Paper Immunostrips for Blood Typing.

Novel high-performance biosensing devices, based on a microporous cellulose matrix, have been of great interest due to their high sensitivity, low cost, and simple operation. Herein, we report on the design and testing of portable paper-based immunostrips (IMS) for in-field blood typing in emergencies requiring blood transfusion. Cellulose fibrils of a paper membrane were functionalized with antibodies via supramolecular interactions. The formation of hydrogen bonds between IgM pentamer and cellulose fibers was corroborated using quantum mechanical calculations with a model cellulose chain and a representative amino acid sequence. In the proposed immunostrips, paper with a pore size of 3 µm dia. was used to enable functionalization of its channels with antibody molecules while blocking the red blood cells (RBC) from channel entering. Under the optimized test conditions, all blood types of AB0 and Rh system could be determined by naked eye examination, requiring only a small blood sample (3.5 µL). The durability of IgM immunostrips against storing has been tested. A new method of statistical evaluation of digitized blood agglutination images, compatible with a clinical five-level system, has been proposed. Critical parameters of the agglutination process have been established to enable future development of automatic blood typing with machine vision and digital data processing.

Antioxidant Activity of Milk and Dairy Products.

The aim of the study was to present a review of literature data on the antioxidant potential of raw milk and dairy products (milk, fermented products, and cheese) and the possibility to modify its level at the milk production and processing stage. Based on the available reports, it can be concluded that the consumption of products that are a rich source of bioactive components improves the antioxidant status of the organism and reduces the risk of development of many civilization diseases. Milk and dairy products are undoubtedly rich sources of antioxidant compounds. Various methods, in particular, ABTS, FRAP, and DPPH assays, are used for the measurement of the overall antioxidant activity of milk and dairy products. Research indicates differences in the total antioxidant capacity of milk between animal species, which result from the differences in the chemical compositions of their milk. The content of antioxidant components in milk and the antioxidant potential can be modified through animal nutrition (e.g., supplementation of animal diets with various natural additives (herbal mixtures, waste from fruit and vegetable processing)). The antioxidant potential of dairy products is associated with the quality of the raw material as well as the bacterial cultures and natural plant additives used. Antioxidant peptides released during milk fermentation increase the antioxidant capacity of dairy products, and the use of probiotic strains contributes its enhancement. Investigations have shown that the antioxidant activity of dairy products can be enhanced by the addition of plant raw materials or their extracts in the production process. Natural plant additives should therefore be widely used in animal nutrition or as functional additives to dairy products.

Organic versus Conventional Raw Cow Milk as Material for Processing.

Milk, as one of the basic raw materials of animal origin, must be of adequate hygienic and physicochemical quality for processing. The aim of the article was to compare the quality of raw milk from three production systems, intensive, traditional (together referred to as conventional), and organic, as material for processing, as well as the quality of products made from it. Particular attention was focused on hygienic quality (somatic cell count and total bacterial count), physical characteristics (acidity), basic nutritional value (content of dry matter, total protein, casein, fat, and lactose), content of health-promoting substances (whey proteins, fatty acids, vitamins, and minerals), and technological parameters (rennet clotting time, heat stability, and protein-to-fat ratio). Research assessing the quality of organic milk and dairy products is significantly less extensive (if available at all) than for milk from conventional production (intensive and traditional). The available reports indicate that raw milk from organic farms is more valuable, especially in terms of the content of health-promoting compounds, including vitamins, fatty acids, whey proteins, and minerals. This applies to organic dairy products as well, mainly cheese and yoghurt. This is explained by the fact that organic farming requires that animals are kept in the pasture. However, the hygienic quality of the raw milk, and often the products as well, raises some concerns; for this reason, organic milk producers should be supported in this regard, e.g., through consultancy and training in Good Hygienic Practices. Importantly, milk production in the traditional and organic systems is in line with the concept of the European Green Deal.

Gated Resonance Energy Transfer (gRET) Controlled by Programmed Death Protein Ligand 1.

The resonance energy transfer (RET) between an excited fluorescent probe molecule and a plasmonic nanoparticle (AuNP) has been investigated to evaluate the effect of protein molecules on the RET efficiency. We have found that the energy transfer to a functionalized AuNP can be modulated by a sub-monolayer film of programmed death-ligand 1 (PD-L1) protein. The interactions of PD-L1 with AuNP@Cit involve incorporation of the protein in AuNP shell and formation of a submonolayer adsorption film with voids enabling gated surface plasmon resonance energy transfer (SPRET). A model of the gated-RET system based on the protein size, estimated using Fisher-Polikarpov-Craievich density approximation, has been developed and can be utilized for other proteins, with minimum data requirement, as well. The value of the equilibrium constant KL determined for the Langmuir isotherm is high: KL = 1.27 × 108 M-1, enabling highly sensitive control of the gated-RET by PD-L1. Thus, with the gated-RET technique, one can determine PD-L1 within the dynamic range, extending from 1.2 to 50 nM. Moreover, we have found that the Gibbs free energy for PD-L1 binding to AuNP@Cit is -46.26 kJ/mol (-11.05 kcal/mol), indicating a strong adsorption with supramolecular interactions. The proposed gated-RET system, with the fluorescence intensity of the fluorophore probe molecule modulated by plasmonic quenching with AuNP and shielding of energy transfer by the adsorbed PD-L1 can be further developed for determination of PD-L1 in pharmaceutical formulations for immune checkpoint control in cancer therapy.

High-performance modified cellulose paper-based biosensors for medical diagnostics and early cancer screening: A concise review.

A remarkable progress in the development of portable paper-based biosensors (PBBs) and microfluidic paper-based analytical devices (µPADs) has recently been achieved. In these devices, a paper formed of microfibers of cellulose, a carbohydrate biopolymer, offers both an ample space in its micropores for analytical reagents storage and a capillary force to drive liquid samples to a dedicated reaction zone for instantaneous detection of the desired analytes. Owing to the low cost and ultra-high sensitivity, these novel devices have become a promising alternative to traditional advanced analytical instruments and offer great potential for applications in medical emergencies, health diagnostics at points-of-care, and broad early-cancer screening. In this review, we focus particularly on recent important achievements in utilization of cellulose and its modifications in portable sensing devices for biomedical applications. The progress in functionalization of cellulose papers with antibodies, nucleic acids and nanomaterials in PBBs and µPADs, is discussed and critically evaluated.

Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent "On-Off" switching DNA aptamer.

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 µM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.

Toward early cancer detection: Focus on biosensing systems and biosensors for an anti-apoptotic protein survivin and survivin mRNA.

The development of biosensors for cancer biomarkers has recently been expanding rapidly, offering promising biomedical applications of these sensors as highly sensitive, selective, and inexpensive bioanalytical tools that can provide alternative methodology to that afforded by the advanced hyphenated-instrumental techniques. In this review, we focus particularly on the detection of a member of the inhibitor of apoptosis proteins (IAP) family, protein survivin (Sur), a ubiquitous re-organizer of the cell life cycle with the ability to inhibit the apoptosis and induce an enhanced proliferation leading to the unimpeded cancer growth and metastasis. Herein, we critically evaluate the progress in the development of novel biosensing systems and biosensors for the detection of two survivin (Sur) biomarkers: the Sur protein and its messenger RNA (Sur mRNA), including immunosensors, electrochemical piezo- and impedance-sensors, electrochemi-luminescence biosensors, genosensors based on oligonucleotide molecular beacons (MBs) with fluorescent or electrochemical transduction, as well as the microfluidic and related analytical platforms based on solution chemistry. The in-situ applications of survivin biomarkers' detection technologies to equip nanocarriers of the controlled drug delivery systems with MB-based fluorescence imaging capability, apoptosis control, and mitigation of the acquired drug resistance are also presented and critically evaluated. Finally, we turn the attention to the application of biosensors for the analysis of Sur biomarkers in exosomes and circulating tumor cells for a non-invasive liquid biopsy. The prospect of a widespread screening for early cancers, based on inexpensive point-of-care testing using biosensors and multiplex biosensor arrays, as a means of reducing the high cancer fatality rate, is discussed.

New ISE-Based Apparatus for Na+, K+, Cl-, pH and Transepithelial Potential Difference Real-Time Simultaneous Measurements of Ion Transport across Epithelial Cells Monolayer⁻Advantages and Pitfalls.

Cystic Fibrosis (CF) is the most common fatal human genetic disease, which is caused by a defect in an anion channel protein (CFTR) that affects ion and water transport across the epithelium. We devised an apparatus to enable the measurement of concentration changes of sodium, potassium, chloride, pH, and transepithelial potential difference by means of ion-selective electrodes that were placed on both sides of a 16HBE14σ human bronchial epithelial cell line that was grown on a porous support. Using flat miniaturized ISE electrodes allows for reducing the medium volume adjacent to cells to approximately 20 µL and detecting changes in ion concentrations that are caused by transport through the cell layer. In contrast to classic electrochemical measurements, in our experiments neither the calibration of electrodes nor the interpretation of results is simple. The calibration solutions might affect cell physiology, the medium composition might change the direction of actions of the membrane channels and transporters, and water flow that might trigger or cut off the transport pathways accompanies the transport of ions. We found that there is an electroneutral transport of sodium chloride in both directions of the cell monolayer in the isosmotic transepithelial concentration gradient of sodium or chloride ions. The ions and water are transported as an isosmotic solution of 145 mM of NaCl.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon.

The anti-apoptotic protein survivin is one of the most promising cancer biomarkers owing to its high expression in human cancers and rare occurrence in normal adult tissues. In this work, we have investigated the role of supramolecular interactions between a graphene oxide (GO) nanosheet nanocarrier and a survivin molecular beacon (SurMB), functionalized by attaching fluorophore Joe and quencher Dabcyl (SurMB-Joe). Molecular dynamics simulations revealed hydrogen bonding of Joe moiety and Dabcyl to GO carriers that considerably increase the SurMB-GO bonding strength. This was confirmed in experimental work by the reduced fluorescence background in the OFF state, thereby increasing the useful analytical signal range for mRNA detection. A new mechanism of hairpin⁻hairpin interaction of GO@SurMB with target oligonucleotides has been proposed. A low limit of detection, LOD = 16 nM (S/N = 3), has been achieved for complementary tDNA using GO@SurMB-Joe nanocarriers. We have demonstrated an efficient internalization of SurMB-Joe-loaded GO nanocarriers in malignant SW480 cells. The proposed tunability of the bonding strength in the attached motifs for MBs immobilized on nanocarriers, via structural modifications, should be useful in gene delivery systems to enhance the efficacy of gene retention, cell transfection and genomic material survivability in the cellular environment.

Hairpin-Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells.

Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

Supramolecular interactions of oxidative stress biomarker glutathione with fluorone black.

Oxidative stress biomarkers, including glutathione (GSH) and related compounds, are involved in a variety of interactions enabling redox potential maintenance in living cells and protection against radicals. Since the oxidative stress is promoting and, in many cases, inducing serious illnesses, monitoring of GSH levels can aid in diagnostics and disease prevention. Herein, we report on the discovery of the formation of a supramolecular ensemble of GSH with fluorone black (9-phenyl fluorone, FB) which is optically active and enables sensitive determination of GSH by resonance elastic light scattering (RELS). We have found that supramolecular interactions of GSH with FB can be probed with spectroscopic, RELS, and electrochemical methods. Our investigations show that RELS intensity for FB solutions increases with GSH concentration while fluorescence emission of FB is not affected, as quenching begins only above 0.8mM GSH. The UV-Vis difference spectra show a positive peak at 383nm and a negative peak at 458nm, indicating a higher-energy absorbing complex in comparison to the non-bonded FB host. Supramolecular interactions of FB with GSH have also been corroborated by electrochemical measurements involving two configurations of FB-GSH ensembles on electrodes: (i) an inverted orientation on Au-coated quartz crystal piezoelectrode (Au@SG-FB), with strong thiolate bonding to gold, and (ii) a non-inverted orientation on glassy carbon electrode (GCE@FB-GS), with weak π-π stacking attachment and efficient charge mediation through the ensemble. The formation of a supramolecular ensemble with hydrogen bonding has also been confirmed by quantum mechanical calculations. The discovery of supramolecular FB-GSH ensemble formation enables elucidating the mechanisms of strong RELS responses, changes in UV-Vis absorption spectra, and the electrochemical reactivity. Also, it provides new insights to the understanding of the efficient charge-transfer in redox potential homeostasis which is likely based on an intermediate formation of a similar type of supramolecular ensembles.

Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers.

The interactions of fluorescent probes and biomolecules with nanocarriers are of key importance to the emerging targeted drug delivery systems. Graphene oxide nanosheets (GONs) as the nanocarriers offer biocompatibility and robust drug binding capacity. The interactions of GONs with fluorophores lead to strong fluorescence quenching, which may interfere with fluorescence bioimaging and biodetection. Herein, we report on the interactions and energy transfers in a model ternary system: GONs-FITC-ATP, where FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'-triphosphate). We have found that FITC fluorescence is considerably quenched by ATP (the quenching constant KSV = 113 ± 22 M-1). The temperature coefficient of KSV is positive (αT = 4.15 M-1deg-1). The detailed analysis of a model for internal self-quenching of FITC indicates that the temperature dependence of the net quenching efficiency η for the FITC-ATP pair is dominated by FITC internal self-quenching modes with their contribution estimated at 79%. The quenching of FITC by GONs is much stronger (KSV = 598 ± 29 M-1) than that of FITC-ATP and is associated with the formation of supramolecular assemblies bound with hydrogen bonding and π-π stacking interactions. For the analysis of the complex behavior of the ternary system GONs-FITC-ATP, a model of chemisorption of ATP on GONs, with partial blocking of FITC quenching, has been developed. Our results indicate that ATP acts as a moderator for FITC quenching by GONs. The interactions between ATP, FITC, and GONs have been corroborated using molecular dynamics and quantum mechanical calculations.

Mitochondria-based biosensors with piezometric and RELS transduction for potassium uptake and release investigations.

Dysfunctional mitochondria appear to be involved in many diseases through their role in respiration, reactive oxygen species generation, and energy production. To aid in the design of new biosensors based on mitochondria (MT), we have investigated the feasibility of detecting ion fluxes through the MT-membrane K+-ion channels using piezosensors with MTs immobilized either by hydrogen bonding or thin polypyrrole (PPy) binding film. We have demonstrated for the first time that the mitochondria-based piezosensors are able to detect ion fluxes and thus be utilized for drug development aimed at ion channel opener- or inhibitor-function. The quartz crystal resonator responding only to mass changes in the lower part of the MT film, penetrated by the acoustic wave, is able to detect a pronounced cationic dynamics in PPy-bonded MT piezosensors despite of the undoped-PPy preference for pure anion dynamics. The control experiments performed by resonance elastic light scattering (RELS) confirmed MT swelling/shrinking, ion dynamics, and osmotic water transfer in MTs, as well as the effects of exposure to a drug valinomycin at sub-nanomolar concentrations.

DNA Strand Replacement Mechanism in Molecular Beacons Encoded for the Detection of Cancer Biomarkers.

Signaling properties of a fluorescent hairpin oligonucleotide molecular beacon (MB) encoded to recognize protein survivin (Sur) mRNA have been investigated. The process of complementary target binding to SurMB with 20-mer loop sequence is spontaneous, as expected, and characterized by a high affinity constant (K = 2.51 × 10(16) M(-1)). However, the slow kinetics at room temperature makes it highly irreversible. To understand the intricacies of target binding to MB, a detailed kinetic study has been performed to determine the rate constants and activation energy Ea for the reaction at physiological temperature (37 °C). Special attention has been paid to assess the value of Ea in view of reports of negative activation enthalpy for some nucleic acid reactions that would make the target binding even slower at increasing temperatures in a non-Arrhenius process. The target-binding rate constant determined is k = 3.99 × 10(3) M(-1) s(-1) at 37 °C with Ea = 28.7 ± 2.3 kcal/mol (120.2 ± 9.6 kJ/mol) for the temperature range of 23 to 55 °C. The positive high value of Ea is consistent with a kinetically controlled classical Arrhenius process. We hypothesize that the likely contribution to the activation energy barrier comes from the SurMB stem melting (tm = 53.7 ± 0.2 °C), which is a necessary step in the completion of target strand hybridization with the SurMB loop. A low limit of detection (LOD = 2 nM) for target tDNA has been achieved. Small effects of conformational polymorphs of SurMB have been observed on melting curves. Although these polymorphs could potentially cause a negative Ea, their effect on kinetic transients for target binding is negligible. No toehold preceding steps in the mechanism of target binding were identified.

Piezometric biosensors for anti-apoptotic protein survivin based on buried positive-potential barrier and immobilized monoclonal antibodies.

The anti-apoptotic protein survivin (Sur) plays an important role in the regulation of cell division and inducing the chemotherapeutic drug resistance. The Sur protein and its mRNA have recently been studied as cancer biomarkers and potential targets for cancer therapy. In this work, we have focused on the design of immunosensors for the detection of Sur based on buried positive-potential barrier layer structure and anti-survivin antibody. The modification of solid AuQC piezoelectrodes was monitored by recording the resonance frequency shift and electrochemical measurements during each step of the sensor preparation. Our results indicate that the immunosensor with covalently bound monoclonal anti-survivin antibody can detect Sur with the limit of detection, LOD=1.7nM (S/N=3σ). The immunosensor applicability for the analysis of real samples was assessed by testing samples of cell lysate solutions obtained from human astrocytoma (glioblastoma) U-87MG cell line, with the experiments performed using the standard addition method. The good linearity of the calibration curves for PBS and lysate solutions at low Sur concentrations confirm the high specificity of the proposed biosensor and good discrimination against nonspecific interactions with lysate components. The calculations indicate that there is still room to increase the Sur capture capacity for Sur while miniaturizing the sensor. The important advantage of the sensor is that it can be reused by a simple regeneration procedure.

Modulation of Plasmon-Enhanced Resonance Energy Transfer to Gold Nanoparticles by Protein Survivin Channeled-Shell Gating.

The resonance energy transfer (RET) from excited fluorescent probe molecules to plasmonic gold nanoparticles (AuNPs) can be gated by modulating the width of channels (gates) in submonolayer protein shells surrounding AuNPs. We have explored the gated-RET (gRET) processes using an antiapoptotic protein survivin (Sur) as the gating material, citrate-capped gold nanoparticles (AuNP@Cit), and fluorescein isothiocyanate as the fluorescent probe. Despite the electrostatic repulsive forces between these components, a strong modulation of RET efficiency by Sur down to 240 pM (S/N = 3) is possible. Using piezometric measurements, we have confirmed the Sur adsorbability on Cit-coated Au surfaces with monolayer coverage: γSur = 5.4 pmol/cm(2) and Langmuirian adsorption constant KL,Sur = 1.09 × 10(9) M(-1). The AuNP@Cit/Sur stability has been corroborated using resonance elastic light scattering. The quantum mechanical calculations indicate that multiple hydrogen bonding between Cit ligands and -NH3(+), =NH2(+), and -NH2 groups of lysines and arginines of Sur have likely facilitated Sur bonding to nanoparticles. A theoretical model of gated-RET has been developed, enabling predictions of the system behavior. In contrast to the positive slope of the Stern-Volmer quenching dependence (F0/F) = f(QA), a negative slope has been obtained for gRET relationship (F0/F) = f(cP), attributed to the dequenching.