Label-free Optical Detection Research Articles

Sensitive Detection of Exosomal Proteins via a Compact Surface Plasmon Resonance Biosensor for Cancer Diagnosis.

Exosomes are small extracellular vesicles released by cells for cell-cell communication. They play important roles in cancer development, metastasis, and drug resistance. Exosomal proteins have been demonstrated by many studies as promising biomarkers for cancer screening, diagnosis, and monitoring. Among many detection techniques, surface plasmon resonance (SPR) is a highly sensitive, label-free, and real-time optical detection method. Commercial prism-based wavelength/angular-modulated SPR sensors afford high sensitivity and resolution, but their large footprint and high cost limit their adaptability for clinical settings. Recently, a nanoplasmonic exosome (nPLEX) assay was developed to detect exosomal proteins for ovarian cancer diagnosis. However, comparing with conventional SPR biosensors, the broad applications of nanoplasmonic biosensors are limited by the difficult and expensive fabrication of nanostructures. We have developed an intensity-modulated, compact SPR biosensor (25 cm × 10 cm × 25 cm) which uses a conventional SPR sensing mechanism and does not require nanostructure fabrication. Calibration from glycerol showed that the compact SPR biosensor offered sensitivity of 9.258 × 103%/RIU and resolution of 8.311 × 10-6 RIU. We have demonstrated the feasibility of the compact SPR biosensor in lung cancer diagnosis using exosomal epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) as biomarkers. It detected a higher level of exosomal EGFR from A549 nonsmall cell lung cancer (NSCLC) cells than BEAS-2B normal cells. With human serum samples, the compact SPR biosensor detected similar levels of exosomal EGFR in NSCLC patients and normal controls, and higher expression of exosomal PD-L1 in NSCLC patients than normal controls. The compact SPR biosensor showed higher detection sensitivity than ELISA and similar sensing accuracy as ELISA. It is a simple and user-friendly sensing platform, which may serve as an in vitro diagnostic test for cancer.

Label-free optical detection of thrombin using a liquid crystal-based aptasensor

We describe a real-time, label-free aptasensor that utilizes the orientational properties of nematic liquid crystals (LCs) for detection of thrombin. The imaging principle is based on the homeotropic orientation of LC films, which can be influenced by thrombin-specific aptamers and N,N-dimethyl-N-(3-(trimethoxysilyl)propyl)-1-octadecanaminiuchloride (DMOAP) immobilized on glass slides. The interactions between thrombin and its specific aptamer, including ionic and hydrogen bonds, can interfere with the homeotropic alignment of LCs, leading to changes from ‘dark’ to ‘bright’ responses that can be visualized simply using crossed polarizers. We describe the condition for maintaining the homeotropic state of LCs, which are suitable for thrombin-specific aptamer experiments, and reactions between aptamer and several enzymes, including thrombin. This novel simple LC-based aptasensor has not only high specificity, but also high sensitivity (low detection limit of 1 pg/mL (26.7 fM)). We also note the blood coagulation reaction between fibrinogen and thrombin, which confirms that the aptamer-bound thrombin is auto-enzymatic. Our result shows that the sensitivity of the proposed sensor increases (1 pg/mL to 0.1 pg/mL) using fibrinogen.

Detection of ochratoxin A in aptamer assay using total internal reflection ellipsometry

The current work is a continuation of our research targeted the development of novel optical sensing technologies for detection of mycotoxins. The method of (TIRE) was developed in the last decade as a combination of spectroscopic ellisometry and SPR and was proved to be a highly sensitive analytical tool in bio-sensing particularly attractive for detection of low molecular weight analytes, such as mycotoxins. The use of aptamers as highly specific artificial molecular receptors to ochratoxin A (OTA) in conjunction with the method Total Internal Reflection Ellipsometry (TIRE) is reported here for the first time. Our results showed a possibility of label-free optical detection of OTA down to 0.01 ppb in concentration in direct assay with specific aptamer. The kinetics of aptamer/OTA binding was studied with dynamic TIRE spectral measurements and allowed evaluating the affinity constant KD = 1.8 10−8 Mol which is characteristic for highly specific aptamer/OTA binding.

Rapid and label‐free optical detection of individual carbon air pollutant nanoparticulates in biomedical samples

Carbonaceous particle exposure and air pollution in general lead to a multitude of adverse human health effects and pose multiple challenges in terms of exposure, risk and safety assessment. Highly desirable for fast screening are label-free approaches for detecting these particle types in biological or medical context. We report a powerful approach for detecting carbonaceous particles using photothermal pump-probe microscopy, which directly probes their strong light absorption. The principle and reliability of this approach is demonstrated by examining 4 different carbon black (CB) species modeling soot with diameters ranging from 13 to 500 nm. Our results show that the proposed approach is applicable to a large number of CB types as well as black carbon. As the particles show a strong absorption over a wide spectral range as compared to other absorbing species, we can image CB particles almost background free. Our pump-probe approach allows label-free optical detection and unambiguous localization of CB particles in (bio)fluids and 3D cellular environments. In combination with fluorescence microscopy, this method allows for simultaneous colocalization of CB with different cellular components using fluorophores as shown here for human lung fibroblasts. We further demonstrate the versatility of pump-probe detection in a flow cell.

Surface-Enhanced Raman Spectroscopy-Based Label-Free Insulin Detection at Physiological Concentrations for Analysis of Islet Performance.

Label-free optical detection of insulin would allow in vitro assessment of pancreatic cell functions in their natural state and expedite diabetes-related clinical research and treatment; however, no existing method has met these criteria at physiological concentrations. Using spatially uniform 3D gold-nanoparticle sensors, we have demonstrated surface-enhanced Raman sensing of insulin in the secretions from human pancreatic islets under low and high glucose environments without the use of labels such as antibodies or aptamers. Label-free measurements of the islet secretions showed excellent correlation among the ambient glucose levels, secreted insulin concentrations, and measured Raman-emission intensities. When excited at 785 nm, plasmonic hotspots of the densely arranged 3D gold-nanoparticle pillars as well as strong interaction between sulfide linkages of the insulin molecules and the gold nanoparticles produced highly sensitive and reliable insulin measurements down to 100 pM. The sensors exhibited a dynamic range of 100 pM to 50 nM with an estimated detection limit of 35 pM, which covers the reported concentration range of insulin observed in pancreatic cell secretions. The sensitivity of this approach is approximately 4 orders of magnitude greater than previously reported results using label-free optical approaches, and it is much more cost-effective than immunoassay-based insulin detection widely used in clinics and laboratories. These promising results may open up new opportunities for insulin sensing in research and clinical applications.

Synthesis and characterization of a nanostructured porous silicon/carbon dot-hybrid for orthogonal molecular detection

A new hybrid guest–host material consisting of a Fabry–Pérot porous silicon (PSi) thin film, a nanostructured high surface-area matrix, and encapsulated fluorescent carbon quantum dots (C-dots) is described. The hybrid is synthesized by a facile in situ pyrolysis treatment of the carbonaceous precursor incorporated within the nanoscale pores of the inorganic host. The effects of nanoconfinement on the integrity of the C-dots and their optical properties are characterized. We show that the resulting hybrid allows for label-free optical detection of target molecules using two orthogonal modalities, that is, the white-light reflectivity of the PSi matrix and the fluorescence of the confined C-dots, and these two signals can be observed and collected simultaneously. The resulting hybrid system exhibits superior sensing performance in comparison with that of the individual components. Notably, we demonstrate that the confined C-dots exhibit greater sensitivity toward various analytes as well as an improved linear response, thus providing evidence of the impact of the host nanoscale porous scaffold on the optical properties of the C-dots. Moreover, we show that this orthogonal detection scheme increases the dynamic range of the sensor and minimizes false-negative results.

Label-free optical detection of action potential in mammalian neurons.

We describe an optical technique for label-free detection of the action potential in cultured mammalian neurons. Induced morphological changes due to action potential propagation in neurons are optically interrogated with a phase sensitive interferometric technique. Optical recordings composed of signal pulses mirror the electrical spike train activity of individual neurons in a network. The optical pulses are transient nanoscale oscillatory changes in the optical path length of varying peak magnitude and temporal width. Exogenous application of glutamate to cortical neuronal cultures produced coincident increase in the electrical and optical activity; both were blocked by application of a Na-channel blocker, Tetrodotoxin. The observed transient change in optical path length in a single optical pulse is primarily due to physical fluctuations of the neuronal cell membrane mediated by a yet unknown electromechanical transduction phenomenon. Our analysis suggests a traveling surface wave in the neuronal cell membrane is responsible for the measured optical signal pulses.

Label-free optical detection of single enzyme-reactant reactions and associated conformational changes.

Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.

Patternable polymer microstructure for optical biosensing

Introduction: Applications of rapid sensing and detection of biological analytes are growing due to the significant environmental monitoring, health screening, cell growth and bio/chemical sensors. These factors influence the research and development on simple, cheap and sensitive functional biomolecular device. The interest in the label-free optical detection has been increased as the optical waveguide has a direct light interaction with surrounding analytes, easy integration with microfluidic system and the capability to provide specific interaction. Methods: In this work, we demonstrate the potential of microstructure as an optical biosensor. Visible wavelength is utilized because it is commonly used in biological and chemical sensing for both label and label-free sensing. The SU8 polymer microstructures waveguides were fabricated on 3.5 µm oxide. The microstructures are simulated using COMSOL Multiphysics. Simulation was recorded based on refractive index that mimic the bioanalytes solution and biological binding. Then the experimental setup is developed to control the optical component and manipulate the liquid samples. The fabricated devices were characterized by using the end-facet technique. Meanwhile, microfluidic channel system was also constructed in order to inject the liquid sample into the sensor surface. Results: The wavelength shift for microresonator structure approximately 41.2 nm for 0.1 increments of surrounding refractive index. The experiments demonstrate that the shift occurred approximately 22.5 nm. In other hand, simulated Bragg grating structure gained the shift at 63.2 nm. Meanwhile, the experimental results achieve approximately 20.3 nm. Conclusions: Thus, both simulation and experimental results strongly indicate that patternable polymer microstructure has a huge potential for optical biosensing applications at visible region.

Femtomole Detection of Proteins Using a Label-Free Nanostructured Porous Silicon Interferometer for Perspective Ultrasensitive Biosensing.

Nanostructured porous silicon (PS) is a promising material for label-free optical detection of biomolecules, though it currently suffers of limited clinical diagnostic applications due to insufficient sensitivity. In this regard, here we introduce an ultrasensitive and robust signal processing strategy for PS biosensors that relies on the calculation of the average value over wavelength of spectral interferograms, namely IAW, obtained on PS interferometer by subtraction (wavelength by wavelength) of reflection spectra acquired after adsorption of biomolecules inside the nanopores from a reference reflection spectrum recorded in acetate buffer. As a case study, we choose to monitor bovine serum albumin (BSA) unspecific adsorption, which has been often employed in the literature as a model for proof-of-concept studies of perspective biosensing applications. The proposed IAW signal processing strategy enables reliable detection of BSA at concentrations in the range from 150 pM to 15 μM (down to 3 orders of magnitude lower than those targeted in the current literature) using a PS interferometer operating in label-free mode without any amplification strategies, with good sample-to-sample reproducibility over the whole range of tested concentrations (%CV = 16% over 5 replicates) and good signal-to-noise ratio also at the lowest tested concentration (S/N ≈ 4.6 at 150 pM). A detection limit (DL) of 20 pM (20 femtomoles, 1 mL) is estimated from the sigmoidal function best fitting (R(2) = 0.989) IAW experimental data over the whole range of tested concentrations. This is the lowest DL that has been reported in the literature since the seminal paper of Sailor and co-workers (1997) on the use of PS interferometer for biosensing, and lowers of 4 orders of magnitude DL attained with label-free PS interferometers using conventional effective optical thickness (EOT) calculation through reflective interferometric Fourier transform spectroscopy. Accordingly, the IAW signal processing strategy envisage bringing PS optical transduction at the forefront of ultrasensitive label-free biosensing techniques, especially for point-of-care clinical analysis where low analyte concentrations have to be detected in a small amount of biological samples.

Label-free optical detection of age-related and diabetic oxidative damage in human aqueous humors.

In this study, we investigate the biochemical characteristics of oxidative stress in age-related macular degeneration (AMD) and diabetic retinopathy (DR) by analyzing aqueous humors. Nondiabetic cataract aqueous humor was used as the control. The level of oxidative damage was evaluated based on changes in Raman spectral intensity. Seven prominent peaks were detected at 1002, 1043, 1062, 1352, 1419, 1454, and 1656 cm-1 . We proposed four multimodal biomarkers to distinguish these peaks based on the ratios of Raman intensities in two wavelengths, including CHO (C-O stretching or C-O-H bending modes), AG (adenine and guanine), PRO-AG (protein and AG), and PHEα (phenylalanine symmetric ring breath and amide I α-helix) markers. The presence of oxidative damage was detected by CHO and AG markers associated with C-O stretching, C-O-H bending modes in carbohydrates (1043 cm-1 ), and the nucleic acids adenine and guanine (1352 cm-1 ), respectively. DR-related oxidative damage was identified by PRO-AG and PHEα markers associated with adenine, guanine, and protein components (1419 and 1454 cm-1 ) and amide I α-helix protein structure (1656 cm-1 ), respectively. AMD-related oxidative damage was identified by four biomarkers. Four multimodal biomarkers with simple linear threshold values achieved high sensitivity of 100% and high specificity of 100% for classifying oxidative stress-induced AMD and DR diseases. Therefore, Raman-based label-free optical detection is effective for detecting the presence of age-related or diabetic oxidative damage in aqueous humor.

Progresses of Somatostatin Quantification for Clinical Practice

Somatostatin (SST), a cyclic polypeptide, exerts a wide range of biological effects. As a result, decreased SST in plasma or tissues usually plays an important role in pathogenesis of various disorders. Exogenous supplement of SST or its analogues has been applied as an effective medical intervention in many diseases. The quantification of SST in plasma or tissues may provide a more rational application of SST in clinical practice. Currently, several methods for SST quantification have been established. The advantages and drawbacks of these methods are reviewed for clinical selection or further study. Moreover, necessary collection, extraction and preservation of samples for SST quantification are also recommended. Surface plasmon resonance (SPR) is a label-free optical detection technique for measuring biomolecular interactions in real-time. Among the several quantitative methods, an SPR-based immunoassay is suggested as a potential method for real-time SST quantification for clinical practice. Keywords: Enzyme-linked immunosorbent assay, high-performance liquid chromatography, octreotide, surface plasmon resonance, radioimmunoassay, somatostatin.

Recent advances in immunosensor for narcotic drug detection.

Introduction: Immunosensor for illicit drugs have gained immense interest and have found several applications for drug abuse monitoring. This technology has offered a low cost detection of narcotics; thereby, providing a confirmatory platform to compliment the existing analytical methods. Methods: In this minireview, we define the basic concept of transducer for immunosensor development that utilizes antibodies and low molecular mass hapten (opiate) molecules. Results: This article emphasizes on recent advances in immunoanalytical techniques for monitoring of opiate drugs. Our results demonstrate that high quality antibodies can be used for immunosensor development against target analyte with greater sensitivity, specificity and precision than other available analytical methods. Conclusion: In this review we highlight the fundamentals of different transducer technologies and its applications for immunosensor development currently being developed in our laboratory using rapid screening via immunochromatographic kit, label free optical detection via enzyme, fluorescence, gold nanoparticles and carbon nanotubes based immunosensing for sensitive and specific monitoring of opiates.

An integrated system for optical and electrical detection of single molecules/particles inside a solid-state nanopore.

Nanopore techniques have proven to be useful tools for single-molecule detection. The combination of optical detection and ionic current measurements enables a new possibility for the parallel readout of multiple nanopores without complex nanofluidics and embedded electrodes. In this study, we developed a new integrated system for the label-free optical and electrical detection of single molecules based on a metal-coated nanopore. The entire system, containing a dark-field microscopy system and an ultralow current detection system with high temporal resolution, was designed and fabricated. An Au-coated nanopore was used to generate the optical signal. Light scattering from a single Au-coated nanopore was measured under a dark-field microscope. A lab-built ultralow current detection system was designed for the correlated optical and electrical readout. This integrated system might provide more direct and detailed information on single analytes inside the nanopore compared with classical ionic current measurements.

Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays

Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays

CMOS-compatible Si3N4 Waveguides for Optical Biosensing

We present a CMOS-compatible silicon nitride (Si3N4) waveguide technology platform suitable for monolithic co-integration with optoelectronics in the visible and <1.1μm near infrared wavelength region. With an optimized fabrication process employing low-temperature plasma enhanced chemical vapour deposition (PECVD) and reactive ion etching (RIE), propagation losses of 0.86dB/cm were achieved at λ=850nm for wire waveguides with a cross section of 600x250 nm2. As an example of application we show Mach-Zehnder interferometer based label-free optical detection of the S-protein/S-peptide interaction. Moreover, we present experimental steps towards the realisation of multi-channel biosensors using spiral shaped waveguide structures ensuring a small form factor.

Probing solid-state nanopores with light for the detection of unlabeled analytes.

Nanopore sensing has enabled label-free single-molecule measurements on a wide variety of analytes, including DNA, RNA, and protein complexes. Much progress has been made toward biotechnological applications; however, electrically probing the ion current introduces nonideal noise components. Here we further develop a method to couple an ionic current to a photon-by-photon counting of fluorescent signal from Ca2+-sensitive dyes and demonstrate label-free optical detection of biopolymer translocation through solid-state nanopores using TIRF and confocal microscopy. We show that by fine adjustment of the CaCl2 gradient, EGTA concentration, and voltage, the optical signals can be localized to the immediate vicinity of the pore. Consequently, the noise spectral density distribution in the optical signal exhibits a nearly flat distribution throughout the entire frequency range. With the use of high-speed photon counting devices in confocal microscopy and higher photon count rates using stronger light sources, we can improve the signal-to-noise ratio of signal acquisition, while the use of wide-field imaging in TIRF can allow for simultaneous quantitative imaging of large arrays of nanopores.

Label-free optical detection of biomolecular translocation through nanopore arrays.

In recent years, nanopores have emerged as exceptionally promising single-molecule sensors due to their ability to detect biomolecules at subfemtomole levels in a label-free manner. Development of a high-throughput nanopore-based biosensor requires multiplexing of nanopore measurements. Electrical detection, however, poses a challenge, as each nanopore circuit must be electrically independent, which requires complex nanofluidics and embedded electrodes. Here, we present an optical method for simultaneous measurements of the ionic current across an array of solid-state nanopores, requiring no additional fabrication steps. Proof-of-principle experiments are conducted that show simultaneous optical detection and characterization of ssDNA and dsDNA using an array of pores. Through a comparison with electrical measurements, we show that optical measurements are capable of accessing equivalent transmembrane current information.

Biomedical applications of holographic microspectroscopy [invited

The identification and quantification of specific molecules are crucial for studying the pathophysiology of cells, tissues, and organs as well as diagnosis and treatment of diseases. Recent advances in holographic microspectroscopy, based on quantitative phase imaging or optical coherence tomography techniques, show promise for label-free noninvasive optical detection and quantification of specific molecules in living cells and tissues (e.g., hemoglobin protein). To provide important insight into the potential employment of holographic spectroscopy techniques in biological research and for related practical applications, we review the principles of holographic microspectroscopy techniques and highlight recent studies.

Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A

The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1μgmL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields.