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.

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.

Analysis of the Effect of Various Potential Antimicrobial Agents on the Quality of the Unpasteurized Carrot Juice.

Short shelf-life and poor microbial quality of minimally processed foods of plant origin pose a serious problem for the food industry. Novel techniques of minimal treatment combined with disinfection are being researched, and, for fresh juice, the addition of antimicrobial agents appears to be a promising route. In this research, fresh, nonfiltered, unpasteurized carrot juice was mixed with four potential antimicrobials (bourbon vanilla extract, peppermint extract, cannabidiol oil, and grapefruit extract). All four variants and the reference pure carrot juice were analyzed for metapopulational changes, microbial changes, and physicochemical changes. The potential antimicrobials used in the research have improved the overall microbial quality of carrot juice across 4 days of storage. However, it is important to notice that each of the four agents had a different spectrum of effectiveness towards the groups identified in the microflora of carrot juice. Additionally, the antimicrobials have increased the diversity of the carrot juice microbiome but did not prevent the occurrence of pathogenic bacteria. In conclusion, the use of antimicrobial agents such as essential oils or their derivatives may be a promising way of improving the microbial quality and prolonging the shelf-life of minimally processed foods, such as fresh juices, but the technique requires further research.

Temperature, Salinity and Garlic Additive Shape the Microbial Community during Traditional Beetroot Fermentation Process.

Plant-based traditional fermented products are attracting a lot of interest in global markets. An example of them is beetroot leaven, which is valued for its high bioactive compound content. The variety of production recipes and the spontaneous nature of red beet fermentation favor its high diversity. This study aimed to analyze the impact of external factors-temperature, brine salinity, and garlic dose-on the beetroot fermentation and bacterial metapopulation responsible for this process. The research results confirmed the significant influence of the selected and analyzed factors in shaping the leaven physicochemical profile including organic acid profile and betalain content. Analysis of bacterial populations proved the crucial importance of the first 48 h of the fermentation process in establishing a stable metapopulation structure and confirmed that this is a targeted process driven by the effect of the analyzed factors. Lactobacillaceae, Enterobacteriaceae, and Leuconostocaceae were observed to be the core microbiome families of the fermented red beet. Regardless of the impact of the tested factors, the leaven maintained the status of a promising source of probiotic bacteria. The results of this research may be helpful in the development of the regional food sector and in improving the quality and safety of traditionally fermented products such as beetroot leaven.

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.

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.

Effect of Processing Treatment and Modified Atmosphere Packing on Carrot's Microbial Community Structure by Illumina MiSeq Sequencing.

The aim of this study was to analyze the microbiome of carrot (Daucus carota subsp. sativus) subjected to minimal pre-treatment (rinsing in organic acid solution) and packaging in a high-oxygen modified atmosphere, and then stored for 17 days under refrigeration conditions (4 °C). The highest levels of bacteria in the carrot microbiome were characterized, at almost 78%, by bacteria belonging to the Enterobacteriaceae and Pseudomonadaceae families. Rinsing in a solution of ascorbic and citric acids resulted in the improvement of microbiological quality in the first day of storage. However, the use of a high-oxygen modified atmosphere extended the shelf life of the minimally processed product. Compared to carrots stored in air, those stored in high oxygen concentration were characterized by a greater ratio of bacteria belonging to the Serratia and Enterobacter genera, and a lower ratio belonging to the Pseudomonas and Pantoea genera. Moreover, the ß-biodiversity analysis confirmed that the oxygen concentration was the main factor influencing the differentiation of the metabiomes of the stored carrots. The bacterial strains isolated from carrots identified by molecular methods were mostly pathogenic or potentially pathogenic microorganisms. Neither the minimal pre-treatment nor packaging in high-oxygen atmosphere was able to eliminate the threat of pathogenic bacteria emerging in the product.

Water-Induced Tuning of the Emission of Polyaniline LEDs within the NIR to Vis Range.

Tuning of the emission within the near-infrared to visible range is observed in p-toluenesulfonic acid-doped polyaniline light emitting diodes (PANI/PTSA), when water molecules are absorbed by the active material (wet PANI/PTSA). This is a hybrid material that combines a conjugated π-electron system and a proton system, both strongly interacting in close contact with each other. The proton system successfully competes with the electron system in excitation energy consumption (when electrically powered), thanks to the inductive resonance energy transfer from electrons to protons in wet PANI/PTSA at the energy levels of combination of vibrations and overtones in water, with subsequent light emission. Wet PANI/PTSA, in which electrons and protons can be excited parallelly owing to fast energy transfer, may emit light in different ranges (on a competitive basis). This results in intense light emission with a maximum at 750 nm (and the spectrum very similar to that of an excited protonic system in water), which is blue-shifted compared to the initial one at ∼850 nm that is generated by the PANI/PTSA dry sample, when electrically powered.

Effect of Resveratrol Treatment on Human Pancreatic Cancer Cells through Alterations of Bcl-2 Family Members.

Pancreatic cancers are among of the most lethal types of neoplasms, and are mostly detected at an advanced stage. Conventional treatment methods such as chemotherapy or radiotherapy often do not bring the desired therapeutic effects. For this reason, natural compounds are increasingly being used as adjuvants in cancer therapy. Polyphenolic compounds, including resveratrol, are of particular interest. The aim of this study is to analyze the antiproliferative and pro-apoptotic mechanisms of resveratrol on human pancreatic cells. The study was carried out on three human pancreatic cancer cell lines: EPP85-181P, EPP85-181RNOV (mitoxantrone-resistant cells) and AsPC-1, as well as the normal pancreatic cell line H6c7. The cytotoxicity of resveratrol in the tested cell lines was assessed by the colorimetric method (MTT) and the flow cytometry method. Three selected concentrations of the compound (25, 50 and 100 µM) were tested in the experiments during a 48-h incubation. TUNEL and Comet assays, flow cytometry, immunocytochemistry, confocal microscopy, real-time PCR and Western Blot analyses were used to evaluate the pleiotropic effect of resveratrol. The results indicate that resveratrol is likely to be anticarcinogenic by inhibiting human pancreatic cancer cell proliferation. In addition, it affects the levels of Bcl-2 pro- and anti-apoptotic proteins. However, it should be emphasized that the activity of resveratrol was specific for each of the tested cell lines, and the most statistically significant changes were observed in the mitoxantrone-resistant cells.

Cytotoxic and Proapoptotic Effects of Resveratrol in In Vitro Studies on Selected Types of Gastrointestinal Cancers.

Cancer diseases are currently one of the greatest health challenges in clinical medicine worldwide. Classic methods of treatment often lead to numerous side effects, including the development of multidrug resistance. For this reason, increasing hope is being placed on compounds of natural origin, mainly due to their pleiotropic effect on different types of cells, protective effect on normal cells and toxic effect on cancerous ones. The most studied group are the polyphenolic compounds, which include resveratrol. The effectiveness of polyphenols in the treatment and prevention of many diseases, including cancer of various origins, has become the basis of many scientific studies. The anticancer effect of resveratrol has been demonstrated at all stages of the carcinogenesis process. Additionally, whether administered by itself or in combination with cytostatics, it may play a significant role in the process of reversing multidrug resistance. A review of the effects of resveratrol in in vitro conditions proves that it has a stronger or weaker antiproliferative and proapoptotic effect on the cells of certain neoplasms of the gastrointestinal tract. Despite the differences in the effect of this compound on different types of cancer, a similar tendency can be observed especially regarding the correlation between the concentration of the compound and the incubation time on the one hand and the antitumour effect on the other hand. The information included in this review may prove helpful in planning in vivo and clinical studies in the future.

Role of tesmin expression in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the most frequent cause of cancer-associated mortality worldwide. Tesmin (MTL5) is a 60 kDa protein which has cysteine rich motifs, characteristic of metallothioneins. Tesmin expression was first observed in germ cells during spermatogenesis. Increased tesmin expression in NSCLC has been described previously. Minichromosome maintenance proteins (MCMs) serve a critical role in replication and cell cycle progression, i.e. in NSCLC. The aim of the present study was to evaluate the localization and intensity of tesmin, MCM5 and MCM7 protein expression in NSCLC and their association with the clinicopathological data of patients. Archival paraffin blocks of 243 cases of NSCLC and 104 non-cancerous tissue samples from the surgical margin (control) were obtained from patients treated at the Clinic of Thoracic Surgery of Wroclaw Medical University (Wroclaw, Poland) between 2010 and 2016, and were used for tissue microarrays and immunohistochemical (IHC) experiments. Laser capture microdissection was used for the isolation of cancer cells from 36 frozen samples of NSCLC and 8 control samples, and subsequently, MTL5, MCM5 and MCM7 mRNA expression was detected separately by reverse transcription-quantitative PCR. Positive cytoplasmic and nuclear tesmin, as well as nuclear MCM5 and MCM7 IHC expression were observed in 95.1, 83.67, 95.51 and 100% of the NSCLC cases, respectively. MTL5, MCM5 and MCM7 mRNA expression was observed in 91.66% of the cancer cases for all genes. The statistical analysis revealed increased tesmin IHC expression in cancer cells compared with the control. A positive correlation was observed between the IHC expression of nuclear tesmin and MCM5 proteins (r=0.33; P<0.0001) and nuclear tesmin and MCM7 proteins (r=0.315; P<0.0001). In addition, a positive correlation between the mRNA expression levels of MTL5 and MCM5 (r=0.421; P<0.05), MTL5 and MCM7 (r=0.557; P<0.01) was demonstrated. The survival analysis revealed that the presence of IHC cytoplasmic tesmin expression was a positive prognostic marker in NSCLC (P=0.0524). Furthermore, in vitro experiments performed on the NCI-H1703 cell line revealed that silencing of MTL5 mRNA and tesmin caused the downregulation of the expression levels of MCM5 and MCM7 and decreased the number of cells in the G2 phase. A positive association among tesmin, MCM5 and MCM7 could indicate a possible role of tesmin in the proliferation of NSCLC cancer cells.

Pilot Test on Pre-Swim Hygiene as a Factor Limiting Trihalomethane Precursors in Pool Water by Reducing Organic Matter in an Operational Facility.

Pool water must be constantly disinfected. Chlorine compounds used to disinfect pools react with organic substances such as sweat, urine, and personal care products introduced into pool water by users and results in the formation of disinfection byproducts. Trihalomethanes (THM), including chloroform and dissolved organic carbon (DOC) concentrations, were quantified using a two-stage process: determining initial THM and chloroform levels; then searching for a cheap and easy-to-use method to improve water quality. The method proposed here to limit THM and DOC concentrations in water is controlled showering. At three swimming pool facilities, chloroform concentrations (13.8 ± 0.33 µg/L, 15.5 ± 0.44 µg/L, and 13.9 ± 0.06 µg/L) were below the threshold concentration of 30 µg/L. At a fourth facility, however, the chloroform concentration exceeded that threshold (40.7 ± 9.68 µg/L) when showering was not controlled. Those conditions improved after the introduction of a mandatory shower; concentrations of DOC, THMs, and chloroform all decreased. The chloroform concentration decreased to 29.4 ± 3.8 µg/L, the THM concentration was 31.3 ± 3.9 µg/L, and the DOC concentration was 6.09 ± 0.05 mg/L. Pilot tests were carried out at real facilities to determine whether the control of pre-swim hygiene was possible. The introduction of proper pre-swim hygiene limited the concentration of DOC in water and can lead to a healthier environment for everyone attending the swimming facility.

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.

Impact of physical and microbiological parameters on proper indoor air quality in nursery.

Although the evaluation of air quality in the residential and office rooms has been significantly developed in recent decades, the issues associated with securing the air quality requirements in nurseries are still not well recognised. This study presents the results of tests regarding the physical and microbiological properties of air in selected rooms of a nursery, including the alternatively variable way of rooms ventilation. The experiment was conducted in four different rooms from the 20th of November 2017 to the 16th of April 2018. The constant measurements of basic parameters of physical air quality in rooms and outside as well as the measurements of microbiological and particulate matter contaminations were conducted in the chosen days of the analysis. The results have confirmed the unsatisfactory air quality in the rooms dedicated to small children. Modernisation of the ventilation system, from a natural one to the supply-exhaust ventilation, has lead to an improvement of physical property of the air, but it did not significantly improve its microbiological quality. Our research indicates that the controlled air flow, method of cleaning the premise and health condition of the children may have a great influence on the physical and microbiological quality of the air.

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.

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.

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.