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.

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.

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.

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.

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.

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.