Tubular toxicity of proteinuria and the progression of chronic kidney disease.

Proteinuria is a well-established biomarker of chronic kidney disease (CKD) and a risk predictor of associated disease outcomes. Proteinuria is also a driver of CKD progression toward end-stage kidney disease. Toxic effects of filtered proteins on proximal tubular epithelial cells enhance tubular atrophy and interstitial fibrosis. The extent of protein toxicity and the underlying molecular mechanisms responsible for tubular injury during proteinuria remain unclear. Nevertheless, albumin elicits its toxic effects when degraded and reabsorbed by proximal tubular epithelial cells. Overall, healthy kidneys excrete over 1000 individual proteins, which may be potentially harmful to proximal tubular epithelial cells when filtered and/or reabsorbed in excess. Proteinuria can cause kidney damage, inflammation and fibrosis by increasing reactive oxygen species, autophagy dysfunction, lysosomal membrane permeabilization, endoplasmic reticulum stress and complement activation. Here we summarize toxic proteins reported in proteinuria and the current understanding of molecular mechanisms of toxicity of proteins on proximal tubular epithelial cells leading to CKD progression.

Immunoassays: Analytical and Clinical Performance, Challenges, and Perspectives of SERS Detection in Comparison with Fluorescent Spectroscopic Detection.

Immunoassays (IAs) with fluorescence-based detection are already well-established commercialized biosensing methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). Immunoassays with surface-enhanced Raman spectroscopy (SERS) detection have received significant attention from the research community for at least two decades, but so far they still lack a wide clinical commercial application. This review, unlike any other review that we have seen, performs a three-dimensional performance comparison of SERS IAs vs. fluorescence IAs. First, we compared the limit of detection (LOD) as a key performance parameter for 30 fluorescence and 30 SERS-based immunoassays reported in the literature. We also compared the clinical performances of a smaller number of available reports for SERS vs. fluorescence immunoassays (FIAs). We found that the median and geometric average LODs are about 1.5-2 orders of magnitude lower for SERS-based immunoassays in comparison to fluorescence-based immunoassays. For instance, the median LOD for SERS IA is 4.3 × 10-13 M, whereas for FIA, it is 1.5 × 10-11 M. However, there is no significant difference in average relative standard deviation (RSD)-both are about 5-6%. The analysis of sensitivity, selectivity, and accuracy reported for a limited number of the published clinical studies with SERS IA and FIA demonstrates an advantage of SERS IA over FIA, at least in terms of the median value for all three of those parameters. We discussed common and specific challenges to the performances of both SERS IA and FIA, while proposing some solutions to mitigate those challenges for both techniques. These challenges include non-specific protein binding, non-specific interactions in the immunoassays, sometimes insufficient reproducibility, relatively long assay times, photobleaching, etc. Overall, this review may be useful for a large number of researchers who would like to use immunoassays, but particularly for those who would like to make improvements and move forward in both SERS-based IAs and fluorescence-based IAs.

SERS for Detection of Proteinuria: A Comparison of Gold, Silver, Al Tape, and Silicon Substrates for Identification of Elevated Protein Concentration in Urine.

Excessive protein excretion in human urine is an early and sensitive marker of diabetic nephropathy and primary and secondary renal disease. Kidney problems, particularly chronic kidney disease, remain among the few growing causes of mortality in the world. Therefore, it is important to develop an efficient, expressive, and low-cost method for protein determination. Surface enhanced Raman spectroscopy (SERS) methods are potential candidates to achieve these criteria. In this paper, a SERS method was developed to distinguish patients with proteinuria from the healthy group. Commercial gold nanoparticles (AuNPs) with diameters of 60 nm and 100 nm, and silver nanoparticles (AgNPs) with a diameter of 100 nm were tested on the surface of four different substrates including silver and gold films, silicon, and aluminum tape. SERS spectra were acquired from 111 unique human urine samples prepared and measured for each of the seven different nanoparticle plus substrate combinations. Data analysis by the PCA-LDA algorithm and the ROC curves gave results for the diagnostic figures of merits. The best sensitivity, specificity, accuracy, and AUC were 0.91, 0.84, 0.88, and 0.94 for the set with 100 nm Au NPs on the silver substrate, respectively. Among the three metal substrates, the substrate with AuNPs and Al tape performed slightly worse than the other three substrates, and 100 nm gold nanoparticles on average produced better results than 60 nm gold nanoparticles. The 60 nm diameter AuNPs and silicon, which is about one order of magnitude more cost-effective than AuNPs and gold film, showed a relative performance close to the performance of 60 nm AuNPs and Au film (average AUC 0.88 (Si) vs. 0.89 (Au)). This is likely the first reported application of unmodified silicon in SERS substrates applied for direct detection of proteins in any biofluid, particularly in urine. These results position silicon and AuNPs@Si in particular as a perspective SERS substrate for direct urine analysis, including clinical diagnostics of proteinuria.

Aluminum Foil vs. Gold Film: Cost-Effective Substrate in Sandwich SERS Immunoassays of Biomarkers Reveals Potential for Selectivity Improvement.

The first application of aluminum foil (Al F) as a low-cost/high-availability substrate for sandwich immunoassay using surface-enhanced Raman spectroscopy (SERS) is reported. Untreated and unmodified Al F and gold film are used as substrates for sandwich SERS immunoassay to detect tuberculosis biomarker MPT64 and human immunoglobulin (hIgG) in less than 24 h. The limits of detection (LODs) for tuberculosis (TB) biomarker MPT64 on Al foil, obtained with commercial antibodies, are about 1.8-1.9 ng/mL, which is comparable to the best LOD (2.1 ng/mL) reported in the literature for sandwich ELISA, made with fresh in-house antibodies. Not only is Al foil competitive with traditional SERS substrate gold for the sandwich SERS immunoassay in terms of LOD, which is in the range 18-30 pM or less than 1 pmol of human IgG, but it also has a large cost/availability advantage over gold film. Moreover, human IgG assays on Al foil and Si showed better selectivity (by about 30-70% on Al foil and at least eightfold on Si) and a nonspecific response to rat or rabbit IgG, in comparison to the selectivity in assays using gold film.

High Contrast Surface Enhanced Fluorescence of Carbon Dot Labeled Bacteria Cells on Aluminum Foil.

Surface enhanced fluorescence (SEF) is observed with very high contrast (100-200) from single E. coli bacteria cells labeled with Carbon nanodots (CDs), on aluminum foil and aluminum film. Likely, it is the first application of organic CDs in SEF. SEF with 633 nm excitation delivered a much higher contrast than SEF with 532 nm excitation. Contrast is the ratio of the fluorescent intensities of labeled CDs to unlabeled (control) cells. High contrast with CDs is also observed on the gold film, silicon, and glass. Enhancement factor (EF) is the ratio of the signal on the metal substrate to the signal on the glass. Single E. coli cells, labeled with commercial graphene quantum dots (GCDs), demonstrated higher EFs (44 on gold, 35 on Al film), but at least one order of magnitude lower contrast (7-10 on aluminum and gold) than cells labeled with organic CDs. Therefore, organic CDs can be a good choice for cell imaging/labeling, capable of achieving a signal to noise (standard deviation of the control) as high as 700 on Al film. Overall, aluminum foil and film are highlighted as inexpensive but efficient substrates for Metal Enhanced Fluorescence, particularly MEF of bacterial cells stained with CDs.

Development and validation of hybrid Brillouin-Raman spectroscopy for non-contact assessment of mechano-chemical properties of urine proteins as biomarkers of kidney diseases.

BACKGROUND: Proteinuria is a major marker of chronic kidney disease (CKD) progression and the predictor of cardiovascular mortality. The rapid development of renal failure is expected in those patients who have higher level of proteinuria however, some patients may have slow decline of renal function despite lower level of urinary protein excretion. The different mechanical (visco-elastic) and chemical properties, as well as the proteome profiles of urinary proteins might explain their tubular toxicity mechanism. Brillouin light scattering (BLS) and surface enhanced Raman scattering (SERS) spectroscopies are non-contact, laser optical-based techniques providing visco-elastic and chemical property information of probed human biofluids. We proposed to study and compare these properties of urinary proteins using BLS and SERS spectroscopies in nephrotic patient and validate hybrid BLS-SERS spectroscopy in diagnostic of urinary proteins as well as their profiling. The project ultimately aims for the development of an optical spectroscopic sensor for rapid, non-contact monitoring of urine samples from patients in clinical settings. METHODS: BLS and SERS spectroscopies will be used for non-contact assessment of urinary proteins in proteinuric patients and healthy subjects and will be cross-validated by Liquid Chromatography-Mass Spectrometry (LC-MS). Participants will be followed-up during the 1 year and all adverse events such as exacerbation of proteinuria, progression of CKD, complications of nephrotic syndrome, disease relapse rate and inefficacy of treatment regimen will be registered referencing incident dates. Associations between urinary protein profiles (obtained from BLS and SERS as well as LC-MS) and adverse outcomes will be evaluated to identify most unfavored protein profiles. DISCUSSION: This prospective study is focused on the development of non-contact hybrid BLS - SERS sensing tool and its clinical deployment for diagnosis and prognosis of proteinuria. We will identify the most important types of urine proteins based on their visco-elasticity, amino-acid profile and molecular weight responsible for the most severe cases of proteinuria and progressive renal function decline. We will aim for the developed hybrid BLS - SERS sensor, as a new diagnostic & prognostic tool, to be transferred to other biomedical applications. TRIAL REGISTRATION: The trial has been approved by ClinicalTrials.gov (Trial registration ID NCT04311684). The date of registration was March 17, 2020.

Strong Surface Enhanced Florescence of Carbon Dot Labeled Bacteria Cells Observed with High Contrast on Gold Film.

Strong surface (metal) enhanced fluorescence (SEF or MEF) is observed from clusters and single E coli bacteria cells labeled with Carbon nanodots (CDs), which were synthesized from date pits. The enhancement factor (EF) for SEF of the cell clusters were close to 50 for both 533 and 633 nm laser excitation wavelength. Those EFs are ratios of emission peak areas from CD labeled cell clusters on gold film to the peak areas of the same batch cell clusters on glass substrate. SEF with 633 nm excitation performed better than SEF with 532 nm excitation, achieving higher fluorescence intensity and much higher contrast. The contrast as high as 66 for cell clusters on gold film is a ratio of fluorescent emission peak area measured at the CD labeled cell clusters to the fluorescent peak area measured at unlabeled cell clusters (autofluorescence) on the same substrate. The contrast with the background (S/N) or the ratio of fluorescent peak area measured at bacteria cells to area measured at bare substrate was as high as 200. This report may pave a way for the broader application of surface enhanced fluorescence and especially metal enhanced fluorescence imaging of CD labeled cells and other biological objects. Graphical abstract Carbon dots, synthesized from dates, are used for direct staining of E coli cells. Emission fluorescent spectroscopy of those CD labelled cells on gold film and glass, demonstrated enhancement factor about 50 for emission on gold as compared to glass, Excitation at 633 nm appears far superior to excitation at 532 nm in terms of contrast (up to 67) with unlabeled cells /control due to decrease in auto fluorescence of cells. Maximum Signal to noise ratio is 200.

Nanoparticle-nanoparticle vs. nanoparticle-substrate hot spot contributions to the SERS signal: studying Raman labelled monomers, dimers and trimers.

We used a combination of Raman microscopy, AFM and TEM to quantify the influence of dimerization on the surface enhanced Raman spectroscopy (SERS) signal for gold and silver nanoparticles (NPs) modified with Raman reporters and situated on gold, silver, and aluminum films and a silicon wafer. The overall increases in the mean SERS enhancement factor (EF) upon dimerization (up by 43% on average) and trimerisation (up by 96% on average) of AuNPs and AgNPs on the studied metal films are within a factor of two, which is moderate when compared to most theoretical models. However, the maximum ratio of EFs for some dimers to the mean EF of monomers can be as high as 5.5 for AgNPs on a gold substrate. In contrast, for dimerization and trimerization of gold and silver NPs on silicon, the mean EF increases by 1-2 orders of magnitude relative to the mean EF of single NPs. Therefore, hot spots in the interparticle gap between gold nanoparticles rather than hot spots between Au nanoparticles and the substrate dominate SERS enhancement for dimers and trimers on a silicon substrate. However, Raman labeled noble metal nanoparticles on plasmonic metal films generate on average SERS enhancement of the same order of magnitude for both types of hot spot zones (e.g. NP/NP and NP/metal film).

Candidate protein biomarkers in chronic kidney disease: a proteomics study.

Proteinuria poses a substantial risk for the progression of chronic kidney disease (CKD) and its related complications. Kidneys excrete hundreds of individual proteins, some with a potential impact on CKD progression or as a marker of the disease. However, the available data on specific urinary proteins and their relationship with CKD severity remain limited. Therefore, we aimed to investigate the urinary proteome and its association with kidney function in CKD patients and healthy controls. The proteomic analysis of urine samples showed CKD stage-specific differences in the number of detected proteins and the exponentially modified protein abundance index for total protein (p = 0.007). Notably, specific urinary proteins such as B2MG, FETUA, VTDB, and AMBP exhibited robust negative associations with kidney function in CKD patients compared to controls. Also, A1AG2, CD44, CD59, CERU, KNG1, LV39, OSTP, RNAS1, SH3L3, and UROM proteins showed positive associations with kidney function in the entire cohort, while LV39, A1BG, and CERU consistently displayed positive associations in patients compared to controls. This study suggests that specific urinary proteins, which were found to be negatively or positively associated with the kidney function of CKD patients, can serve as markers of dysfunctional or functional kidneys, respectively.

SERS immuno- and apta-assays in biosensing/bio-detection: Performance comparison, clinical applications, challenges.

Surface Enhanced Raman Spectroscopy is increasingly used as a sensitive bioanalytical tool for detection of variety of analytes ranging from viruses and bacteria to cancer biomarkers and toxins, etc. This comprehensive review describes principles of operation and compares the performance of immunoassays and aptamer assays with Surface Enhanced Raman scattering (SERS) detection to each other and to some other bioassay methods, including ELISA and fluorescence assays. Both immuno- and aptamer-based assays are categorized into assay on solid substrates, assays with magnetic nanoparticles and assays in laminar flow or/and strip assays. The best performing and recent examples of assays in each category are described in the text and illustrated in the figures. The average performance, particularly, limit of detection (LOD) for each of those methods reflected in 9 tables of the manuscript and average LODs are calculated and compared. We found out that, on average, there is some advantage in terms of LOD for SERS immunoassays (0.5 pM median LOD of 88 papers) vs SERS aptamer-based assays (1.7 pM median LOD of 51 papers). We also tabulated and analyzed the clinical performance of SERS immune and aptamer assays, where selectivity, specificity, and accuracy are reported, we summarized the best examples. We also reviewed challenges to SERS bioassay performance and real-life application, including non-specific protein binding, nanoparticle aggregation, limited nanotag stability, sometimes, relatively long time to results, etc. The proposed solutions to those challenges are also discussed in the review. Overall, this review may be interesting not only to bioanalytical chemist, but to medical and life science researchers who are interested in improvement of bioanalyte detection and diagnostics.

Raman, Infrared and Brillouin Spectroscopies of Biofluids for Medical Diagnostics and for Detection of Biomarkers.

This review surveys Infrared, Raman/SERS and Brillouin spectroscopies for medical diagnostics and detection of biomarkers in biofluids, that include urine, blood, saliva and other biofluids. These optical sensing techniques are non-contact, noninvasive and relatively rapid, accurate, label-free and affordable. However, those techniques still have to overcome some challenges to be widely adopted in routine clinical diagnostics. This review summarizes and provides insights on recent advancements in research within the field of vibrational spectroscopy for medical diagnostics and its use in detection of many health conditions such as kidney injury, cancers, cardiovascular and infectious diseases. The six comprehensive tables in the review and four tables in supplementary information summarize a few dozen experimental papers in terms of such analytical parameters as limit of detection, range, diagnostic sensitivity and specificity, and other figures of merits. Critical comparison between SERS and FTIR methods of analysis reveals that on average the reported sensitivity for biomarkers in biofluids for SERS vs FTIR is about 103 to 105 times higher, since LOD SERS are lower than LOD FTIR by about this factor. High sensitivity gives SERS an edge in detection of many biomarkers present in biofluids at low concentration (nM and sub nM), which can be particularly advantageous for example in early diagnostics of cancer or viral infections.HighlightsRaman, Infrared spectroscopies use low volume of biofluidic samples, little sample preparation, fast time of analysis and relatively inexpensive instrumentation.Applications of SERS may be a bit more complicated than applications of FTIR (e.g., limited shelf life for nanoparticles and substrates, etc.), but this can be generously compensated by much higher (by several order of magnitude) sensitivity in comparison to FTIR.High sensitivity makes SERS a noninvasive analytical method of choice for detection, quantification and diagnostics of many health conditions, metabolites, and drugs, particularly in diagnostics of cancer, including diagnostics of its early stages.FTIR, particularly ATR-FTIR can be a method of choice for efficient sensing of many biomarkers, present in urine, blood and other biofluids at sufficiently high concentrations (mM and even a few µM)Brillouin scattering spectroscopy detecting visco-elastic properties of probed liquid medium, may also find application in clinical analysis of some biofluids, such as cerebrospinal fluid and urine.

Trends in Application of SERS Substrates beyond Ag and Au, and Their Role in Bioanalysis.

This article compares the applications of traditional gold and silver-based SERS substrates and less conventional (Pd/Pt, Cu, Al, Si-based) SERS substrates, focusing on sensing, biosensing, and clinical analysis. In recent decades plethora of new biosensing and clinical SERS applications have fueled the search for more cost-effective, scalable, and stable substrates since traditional gold and silver-based substrates are quite expensive, prone to corrosion, contamination and non-specific binding, particularly by S-containing compounds. Following that, we briefly described our experimental experience with Si and Al-based SERS substrates and systematically analyzed the literature on SERS on substrate materials such as Pd/Pt, Cu, Al, and Si. We tabulated and discussed figures of merit such as enhancement factor (EF) and limit of detection (LOD) from analytical applications of these substrates. The results of the comparison showed that Pd/Pt substrates are not practical due to their high cost; Cu-based substrates are less stable and produce lower signal enhancement. Si and Al-based substrates showed promising results, particularly in combination with gold and silver nanostructures since they could produce comparable EFs and LODs as conventional substrates. In addition, their stability and relatively low cost make them viable alternatives for gold and silver-based substrates. Finally, this review highlighted and compared the clinical performance of non-traditional SERS substrates and traditional gold and silver SERS substrates. We discovered that if we take the average sensitivity, specificity, and accuracy of clinical SERS assays reported in the literature, those parameters, particularly accuracy (93-94%), are similar for SERS bioassays on AgNP@Al, Si-based, Au-based, and Ag-based substrates. We hope that this review will encourage research into SERS biosensing on aluminum, silicon, and some other substrates. These Al and Si based substrates may respond efficiently to the major challenges to the SERS practical application. For instance, they may be not only less expensive, e.g., Al foil, but also in some cases more selective and sometimes more reproducible, when compared to gold-only or silver-only based SERS substrates. Overall, it may result in a greater diversity of applicable SERS substrates, allowing for better optimization and selection of the SERS substrate for a specific sensing/biosensing or clinical application.

Review of COVID-19 testing and diagnostic methods.

More than six billion tests for COVID-19 has been already performed in the world. The testing for SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) virus and corresponding human antibodies is essential not only for diagnostics and treatment of the infection by medical institutions, but also as a pre-requisite for major semi-normal economic and social activities such as international flights, off line work and study in offices, access to malls, sport and social events. Accuracy, sensitivity, specificity, time to results and cost per test are essential parameters of those tests and even minimal improvement in any of them may have noticeable impact on life in the many countries of the world. We described, analyzed and compared methods of COVID-19 detection, while representing their parameters in 22 tables. Also, we compared test performance of some FDA approved test kits with clinical performance of some non-FDA approved methods just described in scientific literature. RT-PCR still remains a golden standard in detection of the virus, but a pressing need for alternative less expensive, more rapid, point of care methods is evident. Those methods that may eventually get developed to satisfy this need are explained, discussed, quantitatively compared. The review has a bioanalytical chemistry prospective, but it may be interesting for a broader circle of readers who are interested in understanding and improvement of COVID-19 testing, helping eventually to leave COVID-19 pandemic in the past.

Urinary Protein Profiling for Potential Biomarkers of Chronic Kidney Disease: A Pilot Study.

Proteinuria is a risk factor for chronic kidney disease (CKD) progression and associated complications. However, there is insufficient information on individual protein components in urine and the severity of CKD. We aimed to investigate urinary proteomics and its association with proteinuria and kidney function in early-stage CKD and in healthy individuals. A 24 h urine sample of 42 individuals (21-CKD and 21-healthy individuals) was used for mass spectrometry-based proteomics analysis. An exponentially modified protein abundance index (emPAI) was calculated for each protein. Data were analyzed by Mascot software using the SwissProt database and bioinformatics tools. Overall, 298 unique proteins were identified in the cohort; of them, 250 proteins belong to the control group with median (IQR) emPAI 39.1 (19−53) and 142 proteins belong to the CKD group with median (IQR) emPAI 67.8 (49−117). The level of 24 h proteinuria positively correlated with emPAI (r = 0.390, p = 0.011). The emPAI of some urinary proteomics had close positive (ALBU, ZA2G, IGKC) and negative (OSTP, CD59, UROM, KNG1, RNAS1, CD44, AMBP) correlations (r < 0.419, p < 0.001) with 24 h proteinuria levels. Additionally, a few proteins (VTDB, AACT, A1AG2, VTNC, and CD44) significantly correlated with kidney function. In this proteomics study, several urinary proteins correlated with proteinuria and kidney function. Pathway analysis identified subpathways potentially related to early proteinuric CKD, allowing the design of prospective studies that explore their response to therapy and their relationship to long-term outcomes.

Review: Detection and quantification of proteins in human urine.

Extensive medical research showed that patients, with high protein concentration in urine, have various kinds of kidney diseases, referred to as proteinuria. Urinary protein biomarkers are useful for diagnosis of many health conditions - kidney and cardio vascular diseases, cancers, diabetes, infections. This review focuses on the instrumental quantification (electrophoresis, chromatography, immunoassays, mass spectrometry, fluorescence spectroscopy, the infrared spectroscopy, and Raman spectroscopy) of proteins (the most of all albumin) in human urine matrix. Different techniques provide unique information on what constituents of the urine are. Due to complex nature of urine, a separation step by electrophoresis or chromatography are often used for proteomics study of urine. Mass spectrometry is a powerful tool for the discovery and the analysis of biomarkers in urine, however, costs of the analysis are high, especially for quantitative analysis. Immunoassays, which often come with fluorescence detection, are major qualitative and quantitative tools in clinical analysis. While Infrared and Raman spectroscopies do not give extensive information about urine, they could become important tools for the routine clinical diagnostics of kidney problems, due to rapidness and low-cost. Thus, it is important to review all the applicable techniques and methods related to urine analysis. In this review, a brief overview of each technique's principle is introduced. Where applicable, research papers about protein determination in urine are summarized with the main figures of merits, such as the limit of detection, the detectable range, recovery and accuracy, when available.

Detection of RNA viruses from influenza and HIV to Ebola and SARS-CoV-2: a review.

RNA-based viruses likely make up the highest pandemic threat among all known pathogens in about the last 100 years, since the Spanish Flu of 1918 with 50 M deaths up to COVID-19. Nowadays, an efficient and affordable testing strategy for such viruses have become the paramount target for the fields of virology and bioanalytical chemistry. The detection of the viruses (influenza, hepatitis, HIV, Zika, SARS, Ebola, SARS-CoV-2, etc.) and human antibodies to these viruses is described and tabulated in terms of the reported methods of detection, time to results, accuracy and specificity, if they are reported. The review is focused, but not limited to publications in the last decade. Finally, the limits of detection for each representative publication are tabulated by detection methods and discussed. These methods include PCR, lateral flow immunoassays, LAMP-based methods, ELISA, electrochemical methods (e.g., amperometry, voltammetry), fluorescence spectroscopy, AFM, SPR and SERS spectroscopy, silver staining and CRISPR-Cas based methods, bio-barcode detection, and resonance light scattering. The review is likely to be interesting for various scientists, and particularly helpful with information for establishing interdisciplinary research.

How gap distance between gold nanoparticles in dimers and trimers on metallic and non-metallic SERS substrates can impact signal enhancement.

The impact of variation in the interparticle gaps in dimers and trimers of gold nanoparticles (AuNPs), modified with Raman reporter (2-MOTP), on surface-enhanced Raman scattering (SERS) intensity, relative to the SERS intensity of a single AuNP, is investigated in this paper. The dimers, trimers, and single particles are investigated on the surfaces of four substrates: gold (Au), aluminium (Al), silver (Ag) film, and silicon (Si) wafer. The interparticle distance between AuNPs was tuned by selecting mercaptocarboxylic acids of various carbon chain lengths when each acid forms a mixed SAM with 2-MOTP. The SERS signal quantification was accomplished by combining maps of SERS intensity from a Raman microscope, optical microscope images (×100), and maps/images from AFM or SEM. In total, we analysed 1224 SERS nanoantennas (533 dimers, 648 monomers, and 43 trimers). The average interparticle gaps were measured using TEM. We observed inverse exponential trends for the Raman intensity ratio and enhancement factor ratio versus gap distance on all substrates. Gold substrate, followed by silicon, showed the highest Raman intensity ratio (9) and dimer vs. monomer enhancement factor ratio (up to 4.5), in addition to the steepest inverse exponential curve. The results may help find a balance between SERS signal reproducibility and signal intensity that would be beneficial for future agglomerated NPs in SERS measurements. The developed method of 3 to 1 map combination by an increase in image transparency can be used to study structure-activity relationships on various substrates in situ, and it can be applied beyond SERS microscopy.

Review: Applications of surface-enhanced fluorescence (SEF) spectroscopy in bio-detection and biosensing.

Surface-enhanced fluorescence (SEF) is rapidly becoming one of the main spectroscopic techniques for the detection of a variety of biomolecules and biomarkers. The main reasons for this trend are the high sensitivity and selectivity, robustness, and speed of this analytical method. Each year, the number of applications that utilize this phenomenon increases and with each such work, the complexity and novelty of the used substrates, procedures, and analytes rises. To obtain a clearer view of this phenomenon and research area, we decided to combine 76 valuable research articles from a variety of different research groups into this mini-review. We present and describe these works concisely and clearly, with a particular interest in the quantitative parameters of the experiment. These sources are classified according to the nature of the analyte, on the contrary to most reviews, which sort them by substrate nature. This point of view gives us insight into the development of this research area and the consequent increase in the complexity of the analyte nature. Moreover, this type of sorting can show possible future routes for the expansion of this research area. Along with the analytes, we can also pay attention to the substrates used for each situation and how the development of substrates affects the direction of research and subsequently, the choice of an analyte. About 108 sources and several interesting trends in the SEF research area over the past 25 years are discussed in this mini-review.

Silver nanocrescents with infrared plasmonic properties as tunable substrates for surface enhanced infrared absorption spectroscopy.

We exploit the unique infrared plasmonic properties of silver nanocrescents (AgNCs) in preparing tunable substrates for surface enhanced infrared absorption (SEIRA) spectroscopy. Fabrication provides good control over the crescents' structural properties which enables tuning of the localized surface plasmon resonances (LSPRs) from the visible through the infrared (IR) regions of the spectrum. Using AgNCs as uniquely tunable IR LSPR substrates, we demonstrate the impact of spectral tuning on maximizing SEIRA signal enhancements measured for adsorbed alkylthiolates. The AgNCs demonstrate the largest reported area-normalized SEIRA signal enhancements which increase from 7,700 to 46,000 depending on the relative positions of the AgNC's LSPR frequency and the molecular vibration frequency. The SEIRA enhancement increases and the absorption band line shape becomes more asymmetric as the AgNCs' LSPR frequency overlaps more extensively with the frequency of the probed molecular vibration. The tunability of the LSPR properties will enable fundamental SEIRA studies and the development of optimized SEIRA substrates for detection and identification of molecular adsorbates.

In situ microarray fabrication and analysis using a microfluidic flow cell array integrated with surface plasmon resonance microscopy.

Surface Plasmon Resonance Microscopy (SPRM) is a promising label-free analytical tool for the real-time study of biomolecule interactions in a microarray format. However, flow cell design and microarray fabrication have hindered throughput and limited applications of SPRM. Here we report the integration of a microfluidic flow cell array (MFCA) with SPRM enabling in situ microarray fabrication and multichannel analysis of biomolecule probe-target interactions. We demonstrate the use of the MFCA for delivery of sample solutions with continuous flow in 24 channels in parallel for rapid microarray creation and binding analysis while using SPRM for real-time monitoring of these processes. Label-free measurement of antibody-antibody interactions demonstrates the capabilities of the integrated MFCA-SPRM system and establishes the first steps of the development of a high-throughput, label-free immunogenicity assay. After in situ probe antibody immobilization, target antibody binding was monitored in real time in 24 channels simultaneously. The limit of detection for this particular antibody pair is 80 ng/mL which is approximately 6 times lower than the industry recommended immunogenicity assay detection limit. The integrated MFCA-SPRM system is a powerful and versatile combination for a range of array-based analyses, including biomarker screening and drug discovery.