Optical Fluorescence Detection Research Articles

Au-Coated ZnO Surface-Enhanced Raman Scattering (SERS) Substrates: Synthesis, Characterization, and Applications in Exosome Detection

Developing a biomolecular detection method that minimizes photodamage while preserving an environment suitable for biological constituents to maintain their physiological state is expected to drive new diagnostic and mechanistic breakthroughs. In addition, ultra-sensitive diagnostic platforms are needed for rapid and point-of-care technologies for various diseases. Considering this, surface-enhanced Raman scattering (SERS) is proposed as a non-destructive and sensitive approach to address the limitations of fluorescence, electrochemical, and other optical detection techniques. However, to advance the applications of SERS, novel approaches that can enhance the signal of substrate materials are needed to improve reproducibility and costs associated with manufacture and scale-up. Due to their physical properties and synthesis, semiconductor-based nanostructures have gained increasing recognition as SERS substrates; however, low signal enhancements have offset their widespread adoption. To address this limitation and assess the potential for use in biological applications, zinc oxide (ZnO) was coated with different concentrations (0.01–0.1 M) of gold (Au) precursor. When crystal violet (CV) was used as a model target with the synthesized substrates, the highest enhancement was obtained with ZnO coated with 0.05 M Au precursor. This substrate was subsequently applied to differentiate exosomes derived from three cell types to provide insight into their molecular diversity. We anticipate this work will serve as a platform for colloidal hybrid SERS substrates in future bio-sensing applications.

Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-μFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-μFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-μFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-μFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-μFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-μFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.

Band-tunable achromatic metalens based on phase change material.

Achromatic metalens have the potential to significantly reduce the size and complexity of broadband imaging systems. A large variety of achromatic metalens has been proposed and most of them have the fixed achromatic band that cannot be actively modified. However, band-tunable is an important function in practical applications such as fluorescence microscopic imaging and optical detection. Here, we propose a bilayer metalens that can switch achromatic bands by taking the advantage of the high refractive index contrast of Sb2S3 between amorphous and crystalline state. By switching the state of Sb2S3, the achromatic band can be reversibly switched between the red region of visible spectrum (650-830 nm) and the near-infrared spectrum (830-1100 nm). This band-tunable design indicates a novel (to our knowledge) method to solve the problem of achromatic focusing in an ultrabroad band. The metalens have an average focusing efficiency of over 35% and 55% in two bands while maintaining diffraction-limited performance. Moreover, through proper design, we can combine different functionalities in two bands such as combining achromatic focusing and diffractive focusing. The proposed metalens have numerous potential applications in tunable displaying, detecting devices and multifunctional devices.

Au@Ag@ZIF-8 multifunction probe with internally o-phenylenediamine encoding for the colorimetric, fluorescence, and SERS multi-mode optical detection of reactive sulfur species

Visual, highly selective, and sensitive detection of sulfur dioxide (SO2) and its derivatives (SO32-) in vitro/in vivo is of great significance in maintaining cell homeostasis. A new Au@Ag@ZIF-8 optical sensor with o-phenylenediamine (OPD) internal coding surface-enhanced Raman scattering (SERS) tags based on the free radical reaction was synthesized for multi-mode detection of sulfite. First, Co2+ is oxidized to Co3+. Second, the oxidoreduction between SO32- and Co3+ produces SO4-• and Co2+ to complete the cycle. The resulting SO4-• is further reacted with the chromogenic substrate OPD to form the polymer. Due to the UV–vis absorbance of OPD polymer being stronger than that of OPD, but the fluorescence intensity is lower, and their Raman peaks are obviously different, this sensor can be used for the detection of SO32- through colorimetric, fluorescence, and Raman multi-mode. The main advantages are as follows: (1) high selectivity; (2) achieve fast, visual, and highly sensitive detection of SO32-, and the low limit of detection (LOD) is 1 nM; (3) improve the reliability of detection and expands the detection range (1 nM-800 μM); (4) demonstrate the applicability in recognizing SO32- in HeLa cells to provide accurate information on the dynamic distribution of sulfur in cells.

Wide field scanning by optical fluorescence of oral squamous cell carcinoma (SCC): case report

ABSTRACT Optical fluorescence detection aims to identify precursor lesions, little noticeable to the human eye, and oral cancer. Squamous cell carcinoma or spinocellular carcinoma is a malignant neoplasm that affects the mouth more. In this article, two clinical cases are analyzed, treated with the use of two types of equipment, namely: the photoevidenciation by optical fluorescence of light-emitting violet wavelength of 405nm, power of 100mW, which is luminous radiation, not Ionizing and in the adjuvant treatment, we used low-power laser therapy, power 100mW, with two wavelengths of 808nm infrared, for pain relief, and the red 660nm, for oral mucositis. In Photodynamic therapy, the Photosensitizing Chimiolux® (methylene blue) was used to control Candida albicans. From these cases, we discuss how a more assertive diagnostic hypothesis can save a life and save time, resources, and efforts for the correct diagnosis of the pathology compared to a biopsy and histopathology negative for neoplasia. We conclude that optical fluorescence has excellent social relevance due to its potential to help the professional not specialized in the establishment of early diagnosis of oral cancer. Early diagnosis improves the rates of death caused by this carcinoma, which would extend the post-diagnosis survival and decrease the financial and emotional costs for the patient and family.

Exponential Molecular Amplification by H2 O2 -Mediated Autocatalytic Deprotection of Boronic Ester Probes to Redox Cyclers.

Herein, a new molecular autocatalytic reaction scheme based on a H2 O2 -mediated deprotection of a boronate ester probe into a redox cycling compound is described, generating an exponential signal gain in the presence of O2 and a reducing agent or enzyme. For such a purpose, new chemosensing probes built around a naphthoquinone/naphthohydroquinone redox-active core, masked by a self-immolative boronic ester protecting group, were designed. With these probes, typical autocatalytic kinetic traces with characteristic lags and exponential phases were obtained by using either UV/Visible or fluorescence optical detection, or by using electrochemical monitoring. Detection of concentrations as low as 0.5 μm H2 O2 and 0.5 nm of a naphthoquinone derivative were achieved in a relatively short time (<1 h). From kinetic analysis of the two cross-activated catalytic loops associated with the autocatalysis, the key parameters governing the autocatalytic reaction network were determined, indirectly showing that the analytical performances are currently limited by the slow nonspecific self-deprotection of boronate probes. Collectively, the present results demonstrate the potential of this new exponential molecular amplification strategy, which, owing to its generic nature and modularity, is quite promising for coupling to a wide range of bioassays involving H2 O2 or redox cycling compounds, or for use as a new building block in the development of more complex chemical reaction networks.

Sensitive determination of l-hydroxyproline in dairy products by capillary electrophoresis with in-capillary optical fiber light-emitting diode-induced fluorescence detection

A sensitive capillary electrophoretic method with in-capillary optical fiber light-emitting diode-induced fluorescence detection (ICOF-LED-IFD-CE) was developed for determination of l-Hyp and further for identification of hydrolyzed leather proteins in dairy products.

Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.

Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared with other popular techniques for measuring high-affinity protein-protein interactions, such as biosensing or calorimetry, the high size resolution of complexes at picomolar concentrations obtained with SV offers a distinct advantage in sensitivity and flexibility of the application. Here, we present a basic protocol for carrying out fluorescence-detected SV experiments and the determination of the size distribution and affinity of protein-antibody complexes with picomolar KD values. Using an EGFP-nanobody interaction as a model, this protocol describes sample preparation, ultracentrifugation, data acquisition, and data analysis. A variation of the protocol applying traditional absorbance or an interference optical system can be used for protein-protein interactions in the micromolar KD value range. Sedimentation experiments typically take ∼3 h of preparation and 6-12 h of run time, followed by data analysis (typically taking 1-3 h).

QF-SSOP: real-time optical property corrected fluorescence imaging.

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively.

Fluorescence Detected Sedimentation Velocity Analytical Ultracentrifugation for Investigating Affinity and Stoichiometry of Protein Interactions

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is monitoring the migration of particles in free solution with real-time optical detection. It provides powerful methods for studying multi-component systems of biomacromolecules such as protein and DNA. It offers exquisite resolution regarding the size and shape of the components and population of co-existing complexes, and therefore binding energies and stoichiometries for reversible interacting systems can be resolved. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying molecules at low concentration because of the high sensitivity and selectivity of fluorescence. In order to make FDS-SV a robust and quantitative method for studying macromolecules, we have previously developed computational tools to specifically account for the unique data structure posed by the confocal design and other geometric factors. Such developments allow FDS-SV to be applied to high-affinity binding systems at concentrations well into the picomolar regime. Recently we extended FDS-SV to multi-dimensional signal detection by employing photoswitchable fluorescent proteins and taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching. Thus, FDS-SV is an excellent tool for the study of high-affinity protein interactions. We will show applications to protein-protein and protein-nucleic acid interactions with multiple states, quantitatively determining the binding affinity, stoichiometry and cooperativity.

Sensitive determination of sulfonamides in environmental water by capillary electrophoresis coupled with both silvering detection window and in-capillary optical fiber light-emitting diode-induced fluorescence detector.

A new detector, silvering detection window and in-capillary optical fiber light-emitting diode-induced fluorescence detector (SDW-ICOF-LED-IFD), is introduced for capillary electrophoresis (CE). The strategy of the work was that half surface of the detection window was coated with silver mirror, which could reflect the undetected fluorescence to the photomultiplier tube to be detected, consequently enhancing the detection sensitivity. Sulfonamides (SAs) are important antibiotics that achieved great applications in many fields. However, they pose a serious threat on the environment and human health when they enter into the environment. The SDW-ICOF-LED-IFD-CE system was used to determine fluorescein isothiocyanate (FITC)-labeled sulfadoxine (SDM), sulfaguanidine (SGD) and sulfamonomethoxine sodium (SMM-Na) in environmental water. The detection results obtained by the SDW-ICOF-LED-IFD-CE system were compared to those acquired by the CE with in-capillary optical fiber light-emitting diode-induced fluorescence detection (ICOF-LED-IFD-CE). The limits of detection (LODs) of SDW-ICOF-LED-IFD-CE and ICOF-LED-IFD-CE were 1.0-2.0 nM and 2.5-7.7 nM (S/N = 3), respectively. The intraday (n = 6) and interday (n = 6) precision of migration time and corresponding peak area for both types of CE were all less than 0.86% and 3.68%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 92.5-102.9%. The results indicated that the sensitivity of the SDW-ICOF-LED-IFD-CE system was improved, and that its reproducibility and accuracy were satisfactory. It was successfully applied to analyze SAs in environmental water.

Integration of a Droplet-Based Microfluidic System and Silicon Nanoribbon FET Sensor.

We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets.

A New Dimension of Detection in Analytical Ultracentrifugation with Fluorescence Detection using Photoswitchable FPs as Time Domain Probes

Multi-component protein complex formation is of great interest in physiological and biochemical studies as it is ubiquitous in biological systems. Analytical ultracentrifugation (AUC) provides powerful methods for studying such systems. It offers information on size, shape and binding energies for interacting systems from the analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the superb hydrodynamic resolution achieved in sedimentation velocity, co-existing complexes can be identified, even in the presence of impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) for AUC substantially extends capabilities for studying high-affinity protein interactions because of the selectivity of fluorescence high sensitivity, allowing studies at picomolar concentrations. To overcome limitations posed by a single excitation wavelength, in the current study, we employed photoswitchable fluorescent proteins (FP) as probes in FDS-AUC. Taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching during FDS-AUC, a new dimension of detection is created that allows resolving specific FPs in multi-component mixtures. We have developed a computational framework for the analysis of sedimentation data exploiting the new temporal dimension. Experimentally we demonstrated this approach using mixtures of FPs commonly used in imaging, which could be readily distinguished from experimental FDS data. This approach extends FDS-AUC to more complicated protein system with multiple components and allows for quantitative characterization of protein complexes with regard to the binding mechanism and stoichiometry under very low macromolecular concentrations.

Integration of programmable microfluidics and on-chip fluorescence detection for biosensing applications.

We describe the integration of an actively controlled programmable microfluidic sample processor with on-chip optical fluorescence detection to create a single, hybrid sensor system. An array of lifting gate microvalves (automaton) is fabricated with soft lithography, which is reconfigurably joined to a liquid-core, anti-resonant reflecting optical waveguide (ARROW) silicon chip fabricated with conventional microfabrication. In the automaton, various sample handling steps such as mixing, transporting, splitting, isolating, and storing are achieved rapidly and precisely to detect viral nucleic acid targets, while the optofluidic chip provides single particle detection sensitivity using integrated optics. Specifically, an assay for detection of viral nucleic acid targets is implemented. Labeled target nucleic acids are first captured and isolated on magnetic microbeads in the automaton, followed by optical detection of single beads on the ARROW chip. The combination of automated microfluidic sample preparation and highly sensitive optical detection opens possibilities for portable instruments for point-of-use analysis of minute, low concentration biological samples.

Combining single-molecule manipulation and imaging for the study of protein-DNA interactions.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Reservoir spectroscopy of5s5p3P2–5snd3D1,2,3transitions in strontium

We perform spectroscopy on the optical dipole transitions 5s5p $^3$P$_2$ - 5s$n$d $^3$D$_{1,2,3}$, $n \in (5,6)$, for all stable isotopes of atomic strontium. We develop a new spectroscopy scheme, in which atoms in the metastable $^3$P$_2$ state are stored in a reservoir before being probed. The method presented here increases the attained precision and accuracy by two orders of magnitude compared to similar experiments performed in a magneto-optical trap or discharge. We show how the state distribution and velocity spread of atoms in the reservoir can be tailored to increase the spectroscopy performance. The absolute transition frequencies are measured with an accuracy of 2 MHz. The isotope shifts are given to within 200 kHz. We calculate the $A$ and $Q$ parameters for the hyperfine structure of the fermionic isotope at the MHz-level. Furthermore, we investigate the branching ratios of the $^3$D$_{J}$ states into the $^3$P$_{J}$ states and discuss immediate implications on schemes of optical pumping and fluorescence detection.

Analysis of High-Affinity Protein Interactions by Fluorescence Optical Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) offers substantial advantages for studying protein complex formation, providing information on size, shape and binding energies for reversible systems from analysis of the sedimentation profiles of molecular mixtures in free solution. By virtue of the hydrodynamic resolution achieved in sedimentation velocity experiments, multiple co-existing complexes can be identified, even in the presence of impurities and aggregates, but until recently the limited sensitivity of AUC has prevented accurate analysis of high affinity interactions. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying high-affinity protein interactions due to the high sensitivity of fluorescence, but the unique characteristics of the optical system in this instrument require new analytical tools. We have developed computational approaches for analysis of FDS data that provide excellent fits to the observed sedimentation boundaries, correcting for effects intrinsic to the use of confocal laser optics. With these tools we have explored the detection limit of FDS, and have now recorded sedimentation signals with hydrodynamic resolution at low picomolar concentrations. This allows us to analyze sedimentation isotherms for interacting systems with Kds in the pM to nM range. Using different systems with high-affinity interaction we will demonstrate how the FDS-AUC system can be reliably applied to study high-affinity protein self- and hetero-associations at orders of magnitude lower concentrations and with much higher accuracy than previously possible.

Investigating High Affinity Protein Self-Association by Fluorescence Optical Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions and the rigorous characterization of binding stoichiometry and affinities in free solution. With a recently introduced commercial fluorescence optical detection system (FDS) it has gained the potential for applications to be extended to high-affinity interactions. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for the study by FDS-SV.

Analysis of High Affinity Self-Association by Fluorescence Optical Sedimentation Velocity Analytical Ultracentrifugation of Labeled Proteins: Opportunities and Limitations

Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein) was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.