Paper electrochemical immunosensor for the rapid screening of Galectin-3 patients with heart failure.

A paper electrochemical immunosensor for the combined binding and quantification of the heart failure (HF) biomarker Galectin-3 has been developed. The simple design of the new sensor is comprised of paper material that is decorated with gold nanostructures, to maximize its electroactive surface area, and functionalized with target-specific recognition molecules to selectively bind the protein from aqueous solutions. The binding of the protein caused the blockage of the electron flow to the sensor electroactive surface, thus causing its oxidation potential to shift and the corresponding current to reduce quantitatively with the increase in the protein concentration within the working range of 0.5ng/mL-8ng/mL (LOQ-0.5 ng/mL). This novel sensor was able to quantify Galectin-3 concentration in saliva samples from HF patients and healthy controls within 20 min with good reproducibility (RSD = 3.64%), without the need for complex sample processing steps. The electrochemical measurements of the patient samples were cross validated by ELISA where the percent agreement between the two methods was found to be 92.7% (RSD = 7.20%). Therefore, the new paper immunosensor sensor has a strong potential for rapid and cost-effective screening of the Galectin 3 biomarker at points of care, thus supporting the timely diagnosis of heart failure.

Impact of Leucine and Magnesium Stearate on the Physicochemical Properties and Aerosolization Behavior of Wet Milled Inhalable Ibuprofen Microparticles for Developing Dry Powder Inhaler Formulation.

This study investigated the development and characterization of leucine and magnesium stearate (MgSt) embedded wet milled inhalable ibuprofen (IBF) dry powder inhaler (DPI) formulations. IBF microparticles were prepared by a wet milling homogenization process and were characterized by SEM, FTIR, DSC, XRD and TGA. Using a Twin-Stage Impinger (TSI), the in vitro aerosolization of the formulations with and without carrier lactose was studied at a flow rate of 60± 5 L/min and the IBF was determined using a validated HPLC method. The flow properties were determined by the Carr's Index (CI), Hausner Ratio (HR) and Angle of Repose (AR) of the milled IBF with 4-6.25% leucine and leucine containing formulations showed higher flow property than those of formulations without leucine. The fine particle fraction (FPF) of IBF from the prepared formulations was significantly (p = 0.000278) higher (37.1 ± 3.8%) compared to the original drug (FPF 3.7 ± 0.9%) owing to the presence of leucine, which enhanced the aerosolization of the milled IBF particles. Using quantitative phase analysis, the XPRD data revealed the crystallinity and accurate weight percentages of the milled IBF in the formulations. FTIR revealed no changes of the structural integrity of the milled IBF in presence of leucine or MgSt. The presence of 2.5% MgSt in the selected formulations produced the highest solubility (252.8 ± 0.6 µg/mL) of IBF compared to that of unmilled IBF (147.4 ± 1.6 µg/mL). The drug dissolution from all formulations containing 4-6.25% leucine showed 12.2-18.6% drug release in 2.5 min; however, 100% IBF dissolution occurred in 2 h whereas around 50% original and dry milled IBF dissolved in 2 h. The results indicated the successful preparation of inhalable IBF microparticles by the wet milling method and the developed DPI formulations with enhanced aerosolization and solubility due to the presence of leucine may be considered as future IBF formulations for inhalation.

Robust Wet Milling Technique for Producing Micronized Ibuprofen Particles with Improved Solubility and Dissolution.

This study investigated a systematic approach for producing ibuprofen (IBF) particles with leucine by wet milling. Using a high shear homogenizer, the particles size of the IBF was reduced. Prepared IBF microparticles were freeze-dried and characterized by using Mastersizer, SEM, DSC, XRD, ATR-FTIR, and TGA. The drug saturation solubility and in-vitro dissolution performance were carried out in phosphate buffer solution (PBS, pH 7.4) at 37°C temperature and IBF were determined using a validated HPLC method. The wet-milled method reduced the particle size from 71.3 to 1.7 µm. The minimum particle size of IBF was obtained in 0.05% Tween 80 solution homogenized at 17,000 rpm for 15 min. The saturated solubility (168.7 µg/mL) of the micronized IBF particles with leucine showed higher compared to that of the original IBF (147.4 µg/mL) in PBS solution. The prepared IBF particles containing 2.5-6.25% leucine showed significantly higher IBF release (100%) compared to that of original drug particles (55.9%) in 120 min. The excipient leucine played a major role in enhancing the solubility and dissolution profile of the prepared IBF particles probably by the formation of hydrogen bonding. The developed wet milling was an efficient and robust technique for reducing the particle size of IBF and could be a useful method for manufacturing drug particles with enhanced solubility and dissolution.

Application of Mesoporous Silica Nanoparticles in Cancer Therapy and Delivery of Repurposed Anthelmintics for Cancer Therapy.

This review focuses on the biomedical application of mesoporous silica nanoparticles (MSNs), mainly focusing on the therapeutic application of MSNs for cancer treatment and specifically on overcoming the challenges of currently available anthelmintics (e.g., low water solubility) as repurposed drugs for cancer treatment. MSNs, due to their promising features, such as tunable pore size and volume, ability to control the drug release, and ability to convert the crystalline state of drugs to an amorphous state, are appropriate carriers for drug delivery with the improved solubility of hydrophobic drugs. The biomedical applications of MSNs can be further improved by the development of MSN-based multimodal anticancer therapeutics (e.g., photosensitizer-, photothermal-, and chemotherapeutics-modified MSNs) and chemical modifications, such as poly ethyleneglycol (PEG)ylation. In this review, various applications of MSNs (photodynamic and sonodynamic therapies, chemotherapy, radiation therapy, gene therapy, immunotherapy) and, in particular, as the carrier of anthelmintics for cancer therapy have been discussed. Additionally, the issues related to the safety of these nanoparticles have been deeply discussed. According to the findings of this literature review, the applications of MSN nanosystems for cancer therapy are a promising approach to improving the efficacy of the diagnostic and chemotherapeutic agents. Moreover, the MSN systems seem to be an efficient strategy to further help to decrease treatment costs by reducing the drug dose.

Towards Label-free detection of viral disease agents through their cell surface proteins: Rapid screening SARS-CoV-2 in biological specimens.

Current methods for the screening of viral infections in clinical settings, such as reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), are expensive, time-consuming, require trained personnel and sophisticated instruments. Therefore, novel sensors that can save time and cost are required specially in remote areas and developing countries that may lack the advanced scientific infrastructure for this task. In this work, we present a sensitive, and highly specific biosensing approach for the detection of harmful viruses that have cysteine residues within the structure of their cell surface proteins. We utilized new method for the rapid screening of SARS-CoV-2 virus in biological fluids through its S1 protein by surface enhanced Raman spectroscopy (SERS). The protein is captured from aqueous solutions and biological specimens using a target-specific extractor substrate. The structure of the purified protein is then modified to convert it into a bio-thiol by breaking the disulfide bonds and freeing up the sulfhydryl (SH) groups of the cysteine residues. The formed biothiol chemisorbs favourably onto a highly sensitive plasmonic sensor and probed by a handheld Raman device in few seconds. The new method was used to screen the S1 protein in aqueous medium, spiked human blood plasma, mucus, and saliva samples down to 150 fg/L. The label-free SERS biosensing method has strong potential for the fingerprint identification many viruses (e.g. the human immunodeficiency virus, the human polyomavirus, the human papilloma virus, the adeno associated viruses, the enteroviruses) through the cysteine residues of their capsid proteins. The new method can be applied at points of care (POC) in remote areas and developing countries lacking sophisticated scientific infrastructure.

A paper-based optical sensor for the screening of viruses through the cysteine residues of their surface proteins: A proof of concept on the detection of coronavirus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health. Current methods such as reverse transcription polymerase chain reaction (qRT-PCR) are complex, expensive, and time-consuming. Rapid, and simple screening methods for the detection of SARS-CoV-2 are critically required to fight the current pandemic. In this work we present a proof of concept for, a simple optical sensing method for the screening of SARS-CoV-2 through its spike protein subunit S1. The method utilizes a target-specific extractor chip to bind the protein from the biological specimens. The disulfide bonds of the protein are then reduced into a biothiol with sulfhydryl (SH) groups that react with a blue-colored benzothiazole azo dye-Hg complex (BAN-Hg) and causes the spontaneous change of its blue color to pink which is observable by the naked eye. A linear relationship between the intensity of the pink color and the logarithm of reduced S1 protein concentration was found within the working range 130 ng.mL-1-1.3 pg mL-1. The lowest limit of detection (LOD) of the assay was 130 fg mL-1. A paper based optical sensor was fabricated by loading the BAN-Hg sensor onto filter paper and used to screen the S1 protein in spiked saliva and patients' nasopharyngeal swabs. The results obtained by the paper sensor corroborated with those obtained by qRT-PCR. The new paper-based sensing method can be extended to the screening of many viruses (e.g. the human immunodeficiency virus, the human polyomavirus, the human papilloma virus, the adeno associated viruses, the enteroviruses) through the cysteine residues of their capsid proteins. The new method has strong potential for screening viruses at pathology labs and in remote areas that lacks advanced scientific infrastructure. Further clinical studies are warranted to validate the new sensing method.

ß-Lactoglobulin-Modified Mesoporous Silica Nanoparticles: A Promising Carrier for the Targeted Delivery of Fenbendazole into Prostate Cancer Cells.

The clinical utilization of fenbendazole (FBZ) as a potential anticancer drug has been limited due to its low water solubility, which causes its low bioavailability. The development of a drug nanoformulation that includes the solubilizing agent as a drug carrier can improve solubility and bioavailability. In this study, Mobil Composition of Matter Number 48 (MCM-48) nanoparticles were synthesized and functionalized with succinylated ß-lactoglobulin (BLG) to prevent early-burst drug release. The BLG-modified amine-functionalized MCM-48 (MCM-BLG) nanoparticles were loaded with FBZ to produce the drug nanoformulation (FBZ-MCM-BLG) and improved the water solubility and, consequently, its anticancer effects against human prostate cancer PC-3 cells. The prepared FBZ-MCM-BLG was characterized in terms of size, zeta potential, drug loading capacity, morphology, thermal and chemical analyses, drug release, cellular uptake, cell viability, cell proliferation, production of reactive oxygen species (ROS), and cell migration. The results demonstrated that the FBZ-MCM-BLG nanoparticles have a spherical morphology with a size and zeta potential of 369 ± 28 nm and 28 ± 0.4 mV, respectively. The drug loading efficiency of the new nanoformulation was 19%. The release of FBZ was pH-dependent; a maximum cumulative release of about 76 and 62% in 12 h and a burst release of 53 and 38% in the first 0.5 h was observed at pH 1.2 and 6.8, respectively. The prepared FBZ-MCM-BLG formulation demonstrated higher cytotoxicity effects against PC-3 cells by 5.6- and 1.8-fold, respectively, when compared to FBZ and FBZ-MCM nanoparticles. The new formulation also increased the production of ROS by 1.6- and 1.2-fold and inhibited the migration of PC-3 cells when compared to the FBZ and FBZ-MCM nanoparticles, respectively. Overall, FBZ-MCM-BLG nanoparticles improved FBZ delivery to PC-3 cells and have the potential to be evaluated for the treatment of prostate cancer following a comprehensive in vivo study.

Development of Thiabendazole-Loaded Mesoporous Silica Nanoparticles for Cancer Therapy.

Thiabendazole (TBZ) is an anthelmintic drug currently studied for anticancer purposes. However, due to its low solubility, its biomedical application has been limited. Using mesoporous silica nanoparticles (MSNPs), such as Mobil Composition of Matter Number 41 (MCM-41), as a drug carrier, is a promising approach to improve the solubility of low water-soluble drugs. In the present work, we aim to develop TBZ-loaded MCM-41 (TBZ MCM-41) nanoparticles to improve the solubility and the therapeutic efficacy of TBZ against prostate cancer PC-3 cells. TBZ MCM-41 nanoparticles were synthesized with a size of 215.9 ± 0.07 nm, a spherical shape, a hexagonal array of channels, and a drug loading capacity of 19.1%. The biological effects of the nanoformulation on PC-3 cells were then evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), IncuCyte live-cell imaging system, cell migration, and reactive oxygen species (ROS) assays. The results demonstrated that TBZ was released from MCM-41 nanoparticles in a controlled manner at pH values of 1.2 and 6.8. The cell viability measurements revealed that the TBZ MCM-41 nanoparticles caused a considerable 2.8-fold increase in the cytotoxicity of TBZ (IC50 127.3 and 46 µM for TBZ and TBZ MCM-41 nanoparticles, respectively). The results of the proliferation assay were in agreement with those of the cell viability measurements, where the MCM-41 increased the cytotoxicity of TBZ in a concentration-dependent manner. Also, the TBZ MCM-41 nanoparticles were found to enhance the potency of the drug and inhibit PC-3 cell migration. In addition, the ROS assay confirmed that TBZ MCM-41 nanoparticles were approximately 15% more potent than TBZ to produce ROS. Overall, the results demonstrated that MCM-41 nanoparticles are a promising carrier to improve the therapeutic efficacy of TBZ against PC-3 cells and suggest evaluating the efficacy of the formulation in vivo.

Label-free identification of Erythropoietin isoforms by surface enhanced Raman spectroscopy.

We present a sensitive label-free surface enhanced Raman spectroscopy (SERS) method for the discrimination between the recombinant and endogenous human Erythropoietin (EPO) isoforms. The proposed methodology comprises a lectin-functionalised extractor chip for the extraction of the recombinant human EPO (rhuEPO) and the endogenous EPO (enEPO) from blood plasma. The disulfide bond molecular structure of the purified isoforms was modified to chemisorb the biomolecules onto a SERS substrate in a unified orientation, thus maximizing the reproducibility and sensitivity of the SERS measurements. The acquired SERS spectra of the EPO isoforms showed diagnostic Raman bands that allowed for the discrimination between rhuEPO and enEPO. The method was also used for the SERS quantification of rhuEPO and enEPO down to 0.1 pM and 0.5 pM, respectively. The SERS determination of the protein isoforms was cross validated against ELISA. The new SERS method has strong potential for the rapid screening of rhuEPO doping in athletes and for the therapeutic drug monitoring of rhuEPO treatment in cancer patients.

PEGylated Mesoporous Silica Nanoparticles (MCM-41): A Promising Carrier for the Targeted Delivery of Fenbendazole into Prostrate Cancer Cells.

Low water solubility and thus low bioavailability limit the clinical application of fenbendazole (FBZ) as a potential anticancer drug. Solubilizing agents, such as Mobil Composition of Matter Number 41 (MCM) as a drug carrier, can improve the water solubility of drugs. In this study, PEGylated MCM (PEG-MCM) nanoparticles (NPs) were synthesized and loaded with FBZ (PEG-MCM-FBZ) to improve its solubility and, as a result, its cytotoxicity effect against human prostate cancer PC-3 cells. The loading efficiency of FBZ onto PEG-MCM NPs was 17.2%. The size and zeta potential of PEG-MCM-FBZ NPs were 366.3 ± 6.9 nm and 24.7 ± 0.4 mV, respectively. They had a spherical shape and released the drug in a controlled manner at pH 1.2 and pH 6.2. PEG-MCM-FBZ were found to inhibit the migration of PC-3 cells, increase the cytotoxicity effects of FBZ against PC-3 cells by 3.8-fold, and were more potent by 1.4-fold, when compared to the non-PEGylated NPs. In addition, PEG-MCM-FBZ promoted the production of reactive oxygen species by 1.3- and 1.2-fold, respectively, when compared to FBZ and MCM-FBZ. Overall, the results demonstrate that PEG-MCM-FBZ NPs enhanced FBZ delivery to PC-3 cells; therefore, they have the potential to treat prostate cancer after a comprehensive in vivo study.

Antibody coated conductive polymer for the electrochemical immunosensing of Human Cardiac Troponin I in blood plasma.

Cardiac troponin I (cTnI) is a sensitive biomarker for cardiovascular disease (CVD). Rapid determination of cTnI concentration in blood can greatly reduce the potential of significant heart damage and heart failure. Herein, we demonstrate a new electrochemical immunosensor for selective affinity binding and rapid detection of cTnI in blood plasma by an electrochemical method. A conductive film of "poly 2,5-bis(2-thienyl)3,4-diamine-terthiophene (PDATT)" was deposited onto an Indium Tin Oxide (ITO) electrode using chronoamperometry. Anti-cardiac troponin I antibody was then attached to the two amine (NH2) groups substituted on the central thiophene of terthiophene repeating unit of the polymer chain via amide bond formation. The gaps on the surface of the antibody coated immunosensor were backfilled with bovine serum albumin (BSA) to prevent nonspecific binding of interfering molecules. Differential pulse voltammetry (DPV) was used to determine cTnI upon the formation of cTnI immunocomplex on the sensing surface, appearing a peak at 0.27 V. The response range was 0.01-100 ng mL-1 with limit of quantification down to 0.01 ng mL-1. The developed immunosensor was used to determine cTnI in spiked blood plasma without interference from cardiac troponin T (cTnT). Therefore, this new sensor can be utilised for the detection of cTnI biomarker in pathological laboratories and points of care in less than 15 min.

Inhaled Edoxaban dry powder inhaler formulations: Development, characterization and their effects on the coagulopathy associated with COVID-19 infection.

Herein, we demonstrated the development and characterization of a dry powder inhaler (DPI) formulation of edoxaban (EDX); and investigated the in-vitro anticoagulation effect for the management of pulmonary or cerebral coagulopathy associated with COVID-19 infection. The formulations were prepared by mixing the inhalable micronized drug with a large carrier lactose and dispersibility enhancers, leucine, and magnesium stearate. The drug-excipient interaction was studied using X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) methods. The drug and excipients showed no physical inter particulate interaction. The in-vitro drug aerosolization from the developed formulation was determined by a Twin Stage Impinger (TSI) at a flow rate of 60 ± 5 L /min. The amount of drug deposition was quantified by an established HPLC-UV method. The fine particle fraction (FPF) of EDX API from drug alone formulation was 7%, whereas the formulations with excipients increased dramatically to almost 7-folds up to 47%. The developed DPI formulation of EDX showed a promising in-vitro anticoagulation effect at a very low concentration. This novel DPI formulation of EDX could be a potential and effective inhalation therapy for managing pulmonary venous thromboembolism (VTE) associated with COVID-19 infection. Further studies are warranted to investigate the toxicity and clinical application of the inhaled EDX DPI formulation.

Puerarin dry powder inhaler formulations for pulmonary delivery: Development and characterization.

This study aims at developing and characterizing the puerarin dry powder inhaler (DPI) formulations for pulmonary delivery. The inhalable particles size (<2 µm) was accomplished by micronization and its morphology was examined by scanning electron microscopy (SEM). The puerarin-excipient interaction in powder mixtures was analyzed by using Fourier transform infrared spectroscopy (FTIR), Raman confocal microscopy, X-Ray powder Diffraction (XRD), and differential scanning calorimetry (DSC) methods. Using a Twin stage impinger (TSI), the in-vitro aerosolization of the powder formulations was carried out at a flow rate of 60 L/min and the drug was quantified by employing a validated HPLC method. No significant interactions between the drug and the excipients were observed in the powder formulations. The fine particle fraction (FPF) of the drug alone was 4.2% which has increased five to six-fold for the formulations with aerosolization enhancers. Formulation containing lactose as large carriers produced 32.7% FPF, which further increased with the addition of dispersibility enhancers, leucine and magnesium stearate (40.8% and 41.2%, respectively). The Raman and FTIR techniques are very useful tool for understanding structural integrity and stability of the puerarin in the powder formulations. The puerarin was found to be compatible with the excipients used and the developed DPI formulation may be considered as an efficient formulation for pulmonary delivery for the management of various diseases at a very low dose.

A SERS quenching method for the sensitive determination of insulin.

In this work, we utilise the disulphide bond structure of insulin and a new benzothiazole Raman probe for the detection of human insulin using surface-enhanced Raman spectroscopy (SERS). The disulphide bond structure of the insulin was reduced to generate free sulfhydryl terminal groups. When reacted with benzothiazole-functionalised gold nanoparticles, the reduced protein desorbs the Raman probe and causes its Raman signal intensity to quench. Using this approach, insulin was quantified in the concentration range of 1 × 10-14 -1 × 10-8 M by SERS quenching. The limit of quantification of insulin by the SERS quenching method was found to be 1 × 10-14 M (0.01 pM or 58 pg/L), which satisfies the requirements for monitoring its blood concentration in patients. Because many proteins and peptides have disulphide bonds in their molecular structures, the new SERS quenching method has a strong potential for the rapid determination of ultralow concentrations of proteins in formulations and biological fluids.

Gold-Deposited Nickel Foam as Recyclable Plasmonic Sensor for Therapeutic Drug Monitoring in Blood by Surface-Enhanced Raman Spectroscopy.

A sensitive and recyclable plasmonic nickel foam sensor has been developed for surface-enhanced Raman spectroscopy (SERS). A simple electrochemical method was used to deposit flower-shaped gold nanostructures onto nickel foam substrate. The high packing of the gold nanoflowers onto the nickel foam led to a high enhancement factor (EF) of 1.6 × 1011. The new SERS sensor was utilized for the direct determination of the broad-spectrum ß-lactam carbapenem antibiotic meropenem in human blood plasma down to one pM. The sensor was also used in High Performance Liquid Chromatography (HPLC)-SERS assembly to provide fingerprint identification of meropenem in human blood plasma. Moreover, the SERS measurements were reproducible in aqueous solution and human blood plasma (RSD = 5.5%) and (RSD = 2.86%), respectively at 200 µg/mL (n = 3), and successfully recycled using a simple method, and hence, used for the repeated determination of the drug by SERS. Therefore, the new sensor has a strong potential to be applied for the therapeutic drug monitoring of meropenem at points of care and intensive care units.

Development and characterization of meropenem dry powder inhaler formulation for pulmonary drug delivery.

Meropenem (MPN), a broad spectrum ß-lactam antibiotic, has been increasingly used in the treatment of moderate to severe bacterial infections. However, due to its short plasma half-life and chemical instability in solution form, it has been challenging to use in the intravenous formulation. This study aims to develop and characterize MPN dry powder inhaler (DPI) formulation for pulmonary delivery. The inhalable MPN particles (1-5 µm) were prepared by micronization. Lactose, L-leucine and magnesium stearate (MgSt) were used in the powder formulation as carriers and dispersibility enhancers. The formulations were characterized by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Raman confocal microscopy, X-Ray powder diffraction analysis (PXRD), and differential scanning calorimetry (DSC) methods. The concentration of MPN was determined by using a validated HPLC method. The Fine Particle Fraction (FPF) of meropenem from powder mixtures was determined by a Twin Stage Impinger (TSI) at a flow rate of 60 L/min. The FPF of the original MPN was 1.91% which was significantly increased to 37.5% for the formulations with excipients. No physical interactions between the drug and the excipients observed. This study revealed the potential of a stable meropenem DPI formulation for pulmonary delivery.

Rapid and selective detection of recombinant human erythropoietin in human blood plasma by a sensitive optical sensor.

Recombinant human erythropoietin (rHuEPO) is an important hormone drug that is used to treat several medical conditions. It is also frequently abused by athletes as a performance enhancing agent at sporting events. The time window of the rHuEPO in blood is short. Therefore, the rapid detection of rHuEPO use/abuse at points of care and in sports requires a selective analytical method and a sensitive sensor. Herein, we present a highly selective method for the rapid detection of rHuEPO in human blood plasma by a sensitive optical sensor. rHuEPO is selectively extracted from human blood plasma by a target-specific extractor chip and converted into a biothiol by reducing its disulfide bond structure. The formed biothiol reacts with a water soluble (E)-1-((6-methoxybenzo[d]thiazole-2-yl)diazenyl)naphthalene-2,6-diolHg(ii) (BAN-Hg) optical sensor and causes its rapid decomposition. This leads to a rapid change in the sensor color from blue to pink that can be observed by the naked eye. The optical sensor was used to quantify rHuEPO in the concentration range 1 × 10-8 M to 1 × 10-12 M by UV-Vis spectroscopy. For the screening of blood plasma, an EPO-specific extractor chip was synthesized and used to selectively extract the protein from the biological matrix prior to its conversion into biothiol and quantification by the optical sensor. Since many proteins have a disulfide bond structure, the new method has strong potential for their rapid sensitive and selective detection by the BAN-Hg sensor and UV-Vis spectroscopy.

Toward Label-Free SERS Detection of Proteins through Their Disulfide Bond Structure.

The molecular structure of many proteins contains disulfide bonds between their cysteine residues. In this work we demonstrate the utilization of the disulfide bond structure of proteins for their label-free determination by surface-enhanced Raman spectroscopy (SERS). The new approach for label-free SERS detection of proteins is demonstrated for human insulin. The protein was selectively extracted from spiked plasma samples using target-specific functionalized nanomaterial. Enzyme-linked immune assay (ELISA) was used to detect insulin in the blood plasma and cross-validate the SERS method. The disulfide bonds in the molecular structure of the protein were chemically reduced and used for their chemisorption onto the gold-coated copper oxide substrate in a unified orientation at a very short distance from the hotspots. The oriented chemisorption of the protein caused significant enhancement to the signal intensity of its Raman vibration modes. This is attributed to the strong short-range electromagnetic and chemical enhancement effects that are experienced by the immobilized protein. Using this approach, label-free and reproducible SERS detection of insulin, down to 10 zM (relative standard deviation [RSD] = 5.52%), was achieved. Sixty-five percent of proteins contain disulfide bonds in their molecular structure. Therefore, the new label-free SERS detection method has strong potential for the determination of ultralow concentrations of proteins at pathology labs and in biology research.

Utilizing the thiol chemistry of biomolecules for the rapid determination of anti-TNF-α drug in blood.

The detection of anti-TNF-α drugs require rapid, selective and sensitive biosensors that can be easily utilised at the point of care. Herein, we demonstrate a new biosensing approach that employs target-specific nanomaterial and label free surface-enhanced Raman spectroscopy (SERS) for the selective extraction and rapid determination of Adalimumab (ADB) in human blood plasma. The new method utilises the tumour necrosis factor (TNF-α) for the fabrication of a target-specific nanomaterial for extraction of ADB. The method also uses the thiol chemistry of the purified antibody drug for its chemisorption onto a gold-coated copper oxide substrate. A handheld Raman spectrophotometer is used for the determination of ADB by label free SERS. The limits of quantification (LOQ) and detection (LOD) of the purified and reduced drug by SERS were 0.10 fM and 0.03 fM respectively. ELISA was used for the cross validation of the SERS quantification of ADB where a 98.8% agreement was found between the two methods. Many proteins have disulfide bonds in their molecular structure. Therefore, the demonstrated biosensing approach can be extended for the rapid screening of other proteins and antibody drugs by developing target-specific extractor nanomaterial and utilizing the disulfide bond structure of the purified biomolecules for their label free SERS detection.

Preconcentration and SERS-based determination of infliximab in blood by using a TNF-α-modified gold-coated copper oxide nanomaterial.

Infliximab (INF) is a chimeric monoclonal immunoglobulin acting against tumor necrosis factor-alpha (TNF-α). The drug is used for the treatment of chronic autoimmune and inflammatory diseases. A target-specific nanomaterial is presented for the extraction of INF from human plasma along with a label-free surface enhanced Raman spectroscopy (SERS) method for its determination using a handheld device. A gold-coated copper oxide chip was functionalized with TNF-α and used to extract the drug from plasma. INF was recovered from the extractor by lowering the pH value to 2.5. The disulfide bond structure of the drug was then reduced and used for its oriented chemisorption onto a gold-coated copper oxide substrate for SERS measurements using the INF-specific band at 936 cm-1. The working range of the SERS method was between 10-7 and 10-14 M of reduced INF. The relative standard deviation (RSD), between three different measurements was 4.2% (intra-day) and 7.1% (inter-day). The quantification and detection limits of the assay (LOQ, LOD) were 0.01 pM and 1.4 fM respectively. The SERS detection was cross-validated against ELISA where 99% agreement was found between the two methods. Graphical abstractSchematic representation of the determination of Infliximab (INF) in blood. A gold coated copper oxide chip was functionalised with tumor necrosis factor (TNF-α) and used to extract INF from blood plasma. The captured INF was released, reduced, chemisorbed onto a second gold-coated copper oxide substrate and screened by surface-enhanced Raman spectroscopy (SERS) using a handheld device.