Rapid SERS Detection of Botulinum Neurotoxin Type A.

Surface-enhanced Raman scattering (SERS) is a powerful technique for decoding of 2-5-component mixes of analytes. Low concentrations of analytes and complex biological media are usually non-decodable with SERS. Recognition molecules, such as antibodies and aptamers, provide an opportunity for a specific binding of ultra-low contents of analyte dissolved in complex biological media. Different approaches have been proposed to provide changes in SERS intensity of an external label upon binding of ultra-low contents of the analytes. In this paper, we propose a SERS-based sensor for the rapid and sensitive detection of botulinum toxin type A. The silver nanoisland SERS substrate was functionalized using an aptamer conjugated with a Raman label. The binding of the target affects the orientation of the label, providing changes in an analytical signal. This trick allowed detecting botulinum toxin type A in a one-stage manner without additional staining with a monotonous dose dependence and a limit of detection of 2.4 ng/mL. The proposed sensor architecture is consistent with the multiarray detection systems for multiplex analyses.

Multiplex Lithographic SERS Aptasensor for Detection of Several Respiratory Viruses in One Pot.

Rapid and reliable techniques for virus identification are required in light of recurring epidemics and pandemics throughout the world. Several techniques have been distributed for testing the flow of patients. Polymerase chain reaction with reverse transcription is a reliable and sensitive, though not rapid, tool. The antibody-based strip is a rapid, though not reliable, and sensitive tool. A set of alternative tools is being developed to meet all the needs of the customer. Surface-enhanced Raman spectroscopy (SERS) provides the possibility of single molecule detection taking several minutes. Here, a multiplex lithographic SERS aptasensor was developed aiming at the detection of several respiratory viruses in one pot within 17 min. The four labeled aptamers were anchored onto the metal surface of four SERS zones; the caught viruses affect the SERS signals of the labels, providing changes in the analytical signals. The sensor was able to decode mixes of SARS-CoV-2 (severe acute respiratory syndrome coronavirus two), influenza A virus, respiratory syncytial virus, and adenovirus within a single experiment through a one-stage recognition process.

Nanoisland SERS-Substrates for Specific Detection and Quantification of Influenza A Virus.

Surface-enhanced Raman spectroscopy (SERS)-based aptasensors for virus determination have attracted a lot of interest recently. This approach provides both specificity due to an aptamer component and a low limit of detection due to signal enhancement by a SERS substrate. The most successful SERS-based aptasensors have a limit of detection (LoD) of 10-100 viral particles per mL (VP/mL) that is advantageous compared to polymerase chain reactions. These characteristics of the sensors require the use of complex substrates. Previously, we described silver nanoisland SERS-substrate with a reproducible and uniform surface, demonstrating high potency for industrial production and a suboptimal LoD of 4 × 105 VP/mL of influenza A virus. Here we describe a study of the sensor morphology, revealing an unexpected mechanism of signal enhancement through the distortion of the nanoisland layer. A novel modification of the aptasensor was proposed with chromium-enhanced adhesion of silver nanoparticles to the surface as well as elimination of the buffer-dependent distortion-triggering steps. As a result, the LoD of the Influenza A virus was decreased to 190 VP/mL, placing the nanoisland SERS-based aptasensors in the rank of the most powerful sensors for viral detection.

Lithographic SERS Aptasensor for Ultrasensitive Detection of SARS-CoV-2 in Biological Fluids.

In this paper, we propose a technology for the rapid and sensitive detection of the whole viral particles of SARS-CoV-2 using double-labeled DNA aptamers as recognition elements together with the SERS method for detecting the optical response. We report on the development of a SERS-aptasensor based on a reproducible lithographic SERS substrate, featuring the combination of high speed, specificity, and ultrasensitive quantitative detection of SARS-CoV-2 virions. The sensor makes it possible to identify SARS-CoV-2 in very low concentrations (the limit of detection was 100 copies/mL), demonstrating a sensitivity level comparable to the existing diagnostic golden standard-the reverse transcription polymerase chain reaction.

SERS-based biosensor with Raman-active external responsive element for rapid determination of adenosine monophosphate.

Phosphorylated adenosine derivatives are important biological molecules with diverse biological functions connected with the energetic balance of the cell, biosynthesis of cell components and regulation of protein activity. Measurement of these compounds provides information about the cell signalling in the body as well as the quantity of microorganisms in the environment. Surface-enhanced Raman spectroscopy (SERS) is an optical method that provides a unique spectrum of a substance at low concentrations. Specificity and limit of detection of SERS-based sensors can be increased drastically using nucleic acid aptamers and Raman-active dyes, respectively. Here we describe an adenosine monophosphate (AMP) biosensor based on AMP-dependent interaction between the well-known DNA aptamer for AMP and a novel Raman-active dye. The SERS intensity of novel Black Hole Quencher-2 (BHQ-2) derivatives was shown to be proportional to the charge of the molecule indicating electrostatic interactions with negatively charged colloidal silver nanoparticles. The novel derivative of BHQ-2 with two amine groups, BHQ-2-(NH2)2, binds an unpaired guanine stacked between guanine-guanine and guanine-adenine mismatches in DNA aptamer-AMP complex with KD = 26 nM as shown by 1H nuclear magnetic resonance, molecular docking and biolayer interferometry. The aptamer is pre-structured by AMP being folded in the conformation favorable for the interaction with BHQ-2-(NH2)2. This specific mechanism of the interaction allows designing of a SERS-based aptasensor with a limit of detection being as low as 3.4 nM of AMP and the dynamic range of nearly 5 orders - from 3.4 nM to 200 µM. The results illustrate a new approach to biosensors where DNA-interacting ligands act as external responsive elements providing an analyte-dependent SERS signal.

A Combination of Membrane Filtration and Raman-Active DNA Ligand Greatly Enhances Sensitivity of SERS-Based Aptasensors for Influenza A Virus.

Biosensors combining the ultrahigh sensitivity of surface-enhanced Raman scattering (SERS) and the specificity of nucleic acid aptamers have recently drawn attention in the detection of respiratory viruses. The most sensitive SERS-based aptasensors allow determining as low as 104 virus particles per mL that is 100-fold lower than any antibody-based lateral flow tests but 10-100-times higher than a routine polymerase chain reaction with reversed transcription (RT-PCR). Sensitivity of RT-PCR has not been achieved in SERS-based aptasensors despite the usage of sophisticated SERS-active substrates. Here, we proposed a novel design of a SERS-based aptasensor with the limit of detection of just 103 particles per ml of the influenza A virus that approaches closely to RT-PCR sensitivity. The sensor utilizes silver nanoparticles with the simplest preparation instead of sophisticated SERS-active surfaces. The analytical signal is provided by a unique Raman-active dye that competes with the virus for the binding to the G-quadruplex core of the aptamer. The aptasensor functions even with aliquots of the biological fluids due to separation of the off-target molecules by pre-filtration through a polymeric membrane. The aptasensor detects influenza viruses in the range of 1·103-5·1010 virus particles per ml.

Aptamer-coated track-etched membranes with a nanostructured silver layer for single virus detection in biological fluids.

Aptasensors based on surface-enhanced Raman spectroscopy (SERS) are of high interest due to the superior specificity and low limit of detection. It is possible to produce stable and cheap SERS-active substrates and portable equipment meeting the requirements of point-of-care devices. Here we combine the membrane filtration and SERS-active substrate in the one pot. This approach allows efficient adsorption of the viruses from the solution onto aptamer-covered silver nanoparticles. Specific determination of the viruses was provided by the aptamer to influenza A virus labeled with the Raman-active label. The SERS-signal from the label was decreased with a descending concentration of the target virus. Even several virus particles in the sample provided an increase in SERS-spectra intensity, requiring only a few minutes for the interaction between the aptamer and the virus. The limit of detection of the aptasensor was as low as 10 viral particles per mL (VP/mL) of influenza A virus or 2 VP/mL per probe. This value overcomes the limit of detection of PCR techniques (∼103 VP/mL). The proposed biosensor is very convenient for point-of-care applications.

SERS-Based Aptasensor for Rapid Quantitative Detection of SARS-CoV-2.

During the COVID-19 pandemic, the development of sensitive and rapid techniques for detection of viruses have become vital. Surface-enhanced Raman scattering (SERS) is an appropriate tool for new techniques due to its high sensitivity. SERS materials modified with short-structured oligonucleotides (DNA aptamers) provide specificity for SERS biosensors. Existing SERS-based aptasensors for rapid virus detection are either inapplicable for quantitative determination or have sophisticated and expensive construction and implementation. In this paper, we provide a SERS-aptasensor based on colloidal solutions which combines rapidity and specificity in quantitative determination of SARS-CoV-2 virus, discriminating it from the other respiratory viruses.

Using the Method of "Optical Biopsy" of Prostatic Tissue to Diagnose Prostate Cancer.

The possibilities of using optical spectroscopy methods in the differential diagnosis of prostate cancer were investigated. Analytical discrimination models of Raman spectra of prostate tissue were constructed by using the projections onto latent structures data analysis(PLS-DA) method for different wavelengths of exciting radiation-532 and 785 nm. These models allowed us to divide the Raman spectra of prostate cancer and the spectra of hyperplasia sites for validation datasets with the accuracy of 70-80%, depending on the specificity value. Meanwhile, for the calibration datasets, the accuracy values reached 100% for the excitation of a laser with a wavelength of 785 nm. Due to the registration of Raman "fingerprints", the main features of cellular metabolism occurring in the tissue of a malignant prostate tumor were confirmed, namely the absence of aerobic glycolysis, over-expression of markers (FASN, SREBP1, stearoyl-CoA desaturase, etc.), and a strong increase in the concentration of cholesterol and its esters, as well as fatty acids and glutamic acid. The presence of an ensemble of Raman peaks with increased intensity, inherent in fatty acid, beta-glucose, glutamic acid, and cholesterol, is a fundamental factor for the identification of prostate cancer.

SERS-Based Colloidal Aptasensors for Quantitative Determination of Influenza Virus.

Development of sensitive techniques for rapid detection of viruses is on a high demand. Surface-enhanced Raman spectroscopy (SERS) is an appropriate tool for new techniques due to its high sensitivity. DNA aptamers are short structured oligonucleotides that can provide specificity for SERS biosensors. Existing SERS-based aptasensors for rapid virus detection had several disadvantages. Some of them lacked possibility of quantitative determination, while others had sophisticated and expensive implementation. In this paper, we provide a new approach that combines rapid specific detection and the possibility of quantitative determination of viruses using the example of influenza A virus.

SERS-Based Biosensors for Virus Determination with Oligonucleotides as Recognition Elements.

Viral infections are among the main causes of morbidity and mortality of humans; sensitive and specific diagnostic methods for the rapid identification of viral pathogens are required. Surface-enhanced Raman spectroscopy (SERS) is one of the most promising techniques for routine analysis due to its excellent sensitivity, simple and low-cost instrumentation and minimal required sample preparation. The outstanding sensitivity of SERS is achieved due to tiny nanostructures which must be assembled before or during the analysis. As for specificity, it may be provided using recognition elements. Antibodies, complimentary nucleic acids and aptamers are the most usable recognition elements for virus identification. Here, SERS-based biosensors for virus identification with oligonucleotides as recognition elements are reviewed, and the potential of these biosensors is discussed.

Spoof plasmons enable giant Raman scattering enhancement in Near-Infrared region.

Exceptionally strong enhancement of the Raman signal exceeding eight orders of magnitude for near-infrared (1064 nm) excitation is demonstrated for an array of dielectric submicron pillars covered by a relatively thick metal layer. The microstructure is designed to support 'spoof' plasmon-polariton excitations with resonant frequencies significantly below the fundamental surface plasmon resonance. Experiments reveal a relatively narrow range of spatial parameters for the optimal resonant scattering enhancement. They include a period close to the excitation wavelength, a specific ratio of the pillar planar size to the period, and optimal heights of both the pillars and the covering silver metal layer. The realized microstructures can be produced by fab-compatible photolithography techniques, and their outstanding sensing possibilities open the venue for the biomedical applications.

Highly sensitive detection of influenza virus with SERS aptasensor.

Highly sensitive and rapid technology of surface enhanced Raman scattering (SERS) was applied to create aptasensors for influenza virus detection. SERS achieves 106-109 times signal amplification, yielding excellent sensitivity, whereas aptamers to hemagglutinin provide a specific recognition of the influenza virus. Aptamer RHA0385 was demonstrated to have essentially broad strain-specificity toward both recombinant hemagglutinins and the whole viruses. To achieve high sensitivity, a sandwich of primary aptamers, influenza virus and secondary aptamers was assembled. Primary aptamers were attached to metal particles of a SERS substrate, and influenza viruses were captured and bound with secondary aptamers labelled with Raman-active molecules. The signal was affected by the concentration of both primary and secondary aptamers. The limit of detection was as low as 1 · 10-4 hemagglutination units per probe as tested for the H3N2 virus (A/England/42/72). Aptamer-based sensors provided recognition of various influenza viral strains, including H1, H3, and H5 hemagglutinin subtypes. Therefore, the aptasensors could be applied for fast and low-cost strain-independent determination of influenza viruses.

Slow plasmon-polaritons in a bilayer metallic structure revealed by the lower-energy resonances of surface-enhanced Raman scattering.

Apart from the main plasmon-polariton resonance of the surface-enhanced Raman scattering (SERS) occurring at 480 - 530 nm, an additional resonance was observed for substrates with two silver layers separated by a dielectric layer which support extra plasmon modes with decreased group velocities. The novel SERS resonance is shifted towards lower energies and has comparable amplitude, its exact energy position being determined by the thickness of the dielectric interlayer. The experimental findings provide a ground for the engineering of SERS-substrates with the spectral position of the additional resonance matched with the photon energy of the pump laser over a fairly wide range of laser wavelengths.