Ultrasensitive and Rapid Detection of Procalcitonin via Waveguide-Enhanced Nanogold-Linked Immunosorbent Assay for Early Sepsis Diagnosis.

Sepsis, a life-threatening inflammatory response, demands economical, accurate, and rapid detection of biomarkers during the critical "golden hour" to reduce the patient mortality rate. Here, we demonstrate a cost-effective waveguide-enhanced nanogold-linked immunosorbent assay (WENLISA) based on nanoplasmonic waveguide biosensors for the rapid and sensitive detection of procalcitonin (PCT), a sepsis-related inflammatory biomarker. To enhance the limit of detection (LOD), we employed sandwich assays using immobilized capture antibodies and detection antibodies conjugated to gold nanoparticles to bind the target analyte, leading to a significant evanescent wave redistribution and strong nanoplasmonic absorption near the waveguide surface. Experimentally, we detected PCT for a wide linear response range of 0.1 pg/mL to 1 ng/mL with a record-low LOD of 48.7 fg/mL (3.74 fM) in 8 min. Furthermore, WENLISA has successfully identified PCT levels in the blood plasma of patients with sepsis and healthy individuals, offering a promising technology for early sepsis diagnosis.

Ultrasensitive amplification-free detection of circulating miRNA via droplet-based processing of SERS tag-miRNA-magnetic nanoparticle sandwich nanocomplexes on a paper-based electrowetting-on-dielectric platform.

MicroRNAs (miRNAs) have emerged as a promising class of biomarkers for early detection of various cancers, including ovarian cancer. However, quantifying miRNAs in human blood samples is challenging owing to the issues of sensitivity and specificity. In this study, hsa-miR-200a-3p of the miR-200a sub-family, which is a biomarker of ovarian cancer, was used as the analyte to demonstrate the analytical capability of an integrated biosensing platform using an extremely sensitive surface-enhanced Raman scattering (SERS) nanotag-nanoaggregate-embedded beads (NAEBs), magnetic nanoparticles (MNPs), a pair of highly specific locked nucleic acid (LNA) probes, and a semi-automated paper-based electrowetting-on-dielectric (pEWOD) device to provide labor-less and thorough sample cleanup and recovery. A sandwich approach where NAEBs are modified by one LNA-1 probe and MNPs are modified by another LNA-2 probe was applied. Then, the target analyte miRNA-200a-3p was introduced to form a sandwich nanocomplex through hybridization with the pair of LNA probes. The pEWOD device was used to achieve short cleanup time and good recovery of the nanocomplex, bringing the total analysis time to less than 30 min. The detection limit of this approach can reach 0.26 fM through SERS detection. The versatility of this method without the need for RNA extraction from clinical samples is expected to have good potential in detecting other miRNAs.

Label-Free Biosensor Based on Particle Plasmon Resonance Coupled with Diffraction Grating Waveguide.

Particle plasmon resonance (PPR), or localized surface plasmon resonance (LSPR), utilizes intrinsic resonance in metal nanoparticles for sensor fabrication. While diffraction grating waveguides monitor bioaffinity adsorption with out-of-plane illumination, integrating them with PPR for biomolecular detection schemes remains underexplored. This study introduces a label-free biosensing platform integrating PPR with a diffraction grating waveguide. Gold nanoparticles are immobilized on a glass slide in contact with a sample, while a UV-assisted embossed diffraction grating is positioned opposite. The setup utilizes diffraction in reflection to detect changes in the environment's refractive index, indicating biomolecular binding at the gold nanoparticle surface. The positional shift of the diffracted beam, measured with varying refractive indices of sucrose solutions, shows a sensitivity of 0.97 mm/RIU at 8 cm from a position-sensitive detector, highlighting enhanced sensitivity due to PPR-diffraction coupling near the gold nanoparticle surface. Furthermore, the sensor achieved a resolution of 3.1 × 10-4 refractive index unit and a detection limit of 4.4 pM for detection of anti-DNP. The sensitivity of the diffracted spot was confirmed using finite element method (FEM) simulations in COMSOL Multiphysics. This study presents a significant advancement in biosensing technology, offering practical solutions for sensitive, rapid, and label-free biomolecule detection.

Competitive Assay for the Ultrasensitive Detection of Organophosphate Pesticides Based on a Fiber-Optic Particle Plasmon Resonance Biosensor and an Acetylcholinesterase Binding Peptide.

An acetylcholinesterase (AChE) binding-based biosensor was developed for the ultrasensitive detection of organophosphate (OP) pesticides. The biosensor integrates the technique based on fiber-optic particle plasmon resonance detection and a synthetic AChE binding peptide conjugated with gold nanoparticles on the optical fiber surface via an AChE competitive binding assay. The OP pesticides present in the solution hinder the binding of AChE to the peptide on the biosensor by competing for the binding sites present in AChE. The limit of detection obtained for parathion using this method was observed to be 0.66 ppt (2.3 pM). This method shows a wide linear dynamic range of 6 orders. Furthermore, the use of the AChE binding peptide in the biosensor can better discriminate OPs against carbamates by using only a single biosensor. The practical application of this method was tested using spiked samples, which yielded good recovery and reproducibility. The spiked sample required minimal pretreatment before analysis; hence, this biosensor may also be used in the field.

Slab waveguide-based particle plasmon resonance optofluidic biosensor for rapid and label-free detection.

An effective bio-sensing platform that would meet the criteria of rapid, simple, and sensitive detection is crucial to translate bench research to clinical applications. However, simultaneously rapid and sensitive biosensing remains challenging for practical biomedical applications. In this study, for the first time, we demonstrate a cost-effective, label-free, real-time, and sensitive slab waveguide-based particle plasmon resonance (WGPPR) biosensor for practical clinical applications. A suspended glass slab waveguide structure with excellent optical confinement properties was designed and fabricated as the biosensor. Gold nanoparticles (AuNPs) were deposited on the top surface of the waveguide layer to significantly enhance the optical near field through the localized surface plasmon resonance (LSPR) effect. When light travels through the waveguide, the change in the local refractive index (RI) near the surface of the AuNPs can be transformed into changes in the intensity of transmitted light, thereby enabling sensitive and real-time detection. The RI sensing experiment shows a good sensor resolution of 1.43 × 10-4 RIU, which represents a 395% enhancement compared to that of the sensor without AuNPs. Through biochemical detection experiments, we measured IgG and determined the detection limit (LOD) at 614 ng mL-1 in ∼4 min, thereby proving the feasibility of the bio-detection sensing functionality. This study demonstrates a new type of WGPPR biosensor, which offers several unique advantages such as simple structure, high sensitivity, and rapid bio-sensing for practical bio-medical sensing applications. The new biosensor also fulfils point-of-care (POC) requirements.

Dual-functional gold-iron oxide core-satellite hybrid nanoparticles for sensitivity enhancement in biosensors via nanoplasmonic and preconcentration effects.

A common strategy to improve the sensitivity of a biosensor for the detection of a low abundance analyte is to preconcentrate the analyte molecules before detection. A dual-functional gold-iron oxide core-satellite hybrid nanoparticle structure is proposed in this work to overcome the drawbacks of traditional sample pretreatment methods and the methods using non-magnetic nanomaterials for sample pretreatment. The new dual-functional hybrid nanoparticle structure can simultaneously serve as a signal reporter of a biorecognition event and a preconcentrator of a target at an extremely low concentration in a nanoplasmonic biosensor. By utilizing a fiber optic nanogold-linked sorbent assay in the fiber optic particle plasmon resonance (FOPPR) biosensor and an arbitrary DNA sequence as a target, we have demonstrated that the use of the new hybrid nanoparticle structure with magnetic preconcentration improves the limit of detection (LOD) for the DNA by 18 times as compared to the same method without magnetic preconcentration, so that the LOD for detecting the DNA can be as low as 2.6 fM. The new hybrid nanoparticle structure is easy to prepare and its use in the high-sensitivity and low-cost FOPPR biosensor provides vast opportunities in point-of-care applications.

MutS protein-based fiber optic particle plasmon resonance biosensor for detecting single nucleotide polymorphisms.

A new biosensing method is presented to detect gene mutation by integrating the MutS protein from bacteria with a fiber optic particle plasmon resonance (FOPPR) sensing system. In this method, the MutS protein is conjugated with gold nanoparticles (AuNPs) deposited on an optical fiber core surface. The target double-stranded DNA containing an A and C mismatched base pair in a sample can be captured by the MutS protein, causing increased absorption of green light launching into the fiber and hence a decrease in transmitted light intensity through the fiber. As the signal change is enhanced through consecutive total internal reflections along the fiber, the limit of detection for an AC mismatch heteroduplex DNA can be as low as 0.49 nM. Because a microfluidic chip is used to contain the optical fiber, the narrow channel width allows an analysis time as short as 15 min. Furthermore, the label-free and real-time nature of the FOPPR sensing system enables determination of binding affinity and kinetics between MutS and single-base mismatched DNA. The method has been validated using a heterozygous PCR sample from a patient to determine the allelic fraction. The obtained allelic fraction of 0.474 reasonably agrees with the expected allelic fraction of 0.5. Therefore, the MutS-functionalized FOPPR sensor may potentially provide a convenient quantitative tool to detect single nucleotide polymorphisms in biological samples with a short analysis time at the point-of-care sites.

Low-cost planar waveguide-based optofluidic sensor for real-time refractive index sensing.

We report on the design, fabrication, and characterization of mass-producible, sensitive, intensity-detection-based planar waveguide sensors for rapid refractive index (RI) sensing; the sensors comprise suspended glass planar waveguides on glass substrates, and are integrated with microfluidic channels. They are facilely and cost-effectively constructed via vacuum-less processes. They yield a high throughput, enabling mass production. The sensors respond to solutions with different RIs via variations in the transmitted optical power due to coupling loss in the sensing region, facilitating real-time and simple RI detection. Experiments yield a good resolution of 5.65 × 10-4 RIU. This work has major implications for several RI-sensing-based applications.

Controlled Silanization: High Molecular Regularity of Functional Thiol Groups on Siloxane Coatings.

A comparative study on deposition and molecular regularity of two organosilanes, i.e., commercially available (3-mercaptopropyl)trimethoxysilane (MPTMS) and newly developed mercaptopropylsilatrane (MPS), was conducted in this work. MPTMS and MPS were applied to modify silicon surfaces to characterize their deposition kinetics, surface morphology, thickness, and elemental composition and the reactivity of thiol end groups based on gold-thiol and thiol-ene chemistries. MPS possesses a tricyclic caged structure and a transannular N → Si dative bond, making it chemically stable and controllable to avoid fast hydrolysis and aggregation in solution. The results indicate that MPS allows faster deposition and better formation of thin and homogeneous films than MPTMS. More importantly, the functional thiol groups on MPS coatings enable immobilization of a large amount of gold nanoparticles and effective thiol-ene photopolymerization with zwitterionic sulfobetaine acrylamide. Postmodification on silanized surfaces with MPS endows excellent plasmonic and antifouling properties, potentially leading to valuable applications to biosensing and biomaterials. The work demonstrated the feasibility and applicability of the functional silatrane molecule for surface silanization in a controlled manner.

A fiber optic nanoplasmonic biosensor for the sensitive detection of ampicillin and its analogs.

A novel optical immunosensor for the screening of ampicillin (Amp) residues has been developed. The biosensor is based on fiber optic particle plasmon resonance detection and uses an enhancement method called as fiber optic nanogold-linked immunosorbent assay (FONLISA) for the sensitive detection of antibiotics. A commercial antibody which had a higher affinity for ampicillin than for other ß-lactam antibiotics was chosen. A surface competitive binding assay was used in which a fixed concentration of antibiotic-conjugated gold nanoparticles (AuNPs) competes with free unlabeled antibiotic molecules to measure the amount of binding with antibody molecules immobilized on an optical fiber. The synthesis of the 11-mercaptoundecanoic acid (MUA)-ampicillin conjugate facilitates the attachment of the Amp molecules to AuNPs via MUA which acts as a linker between them. This AuNP-Amp conjugate was then used for the detection of ß-lactam antibiotics. The practical limit of detection obtained for Amp was 0.74 ppb (7.4 × 10-10 g/mL) which is lower than the recommended maximum residue limit (MRL) for ß-lactams. The method also shows a wide linear range of 4 orders. Its applicability to the determination of ampicillin in spiked milk samples has been demonstrated with good recovery and reproducibility. Graphical abstract.

Integration of a Thermoelectric Heating Unit with Ionic Wind-Induced Droplet Centrifugation Chip to Develop Miniaturized Concentration Device for Rapid Determination of Salmonella on Food Samples Using Antibody-Functionalized SERS Tags.

When a centrifugation-enriched sample of 100 µL containing the surface-enhanced Raman scattering (SERS) tag-bound bacteria (Salmonella in this study) is siphoned onto a glass slide next to an embedded thermoelectric heating chip, such a sessile droplet is quickly evaporated. As the size of the sample droplet is significantly reduced during the heating process, ionic wind streams from a corona discharge needle, stationed above the sample, sweep across the liquid surface to produce centrifugal vortex flow. Tag-bound Salmonella in the sample are then dragged and trapped at the center of droplet bottom. Finally, when the sample is dried, unlike the "coffee ring" effect, the SERS tag-bound Salmonella is concentrated in one small spot to allow sensitive detection of a Raman signal. Compared with our previous electrohydrodynamic concentration device containing only a corona discharge needle, this thermoelectric evaporation-assisted device is more time-effective, with the time of concentrating and drying about 100 µL sample reduced from 2 h to 30 min. Hence, sample throughput can be accelerated with this device for practical use. It is also more sensitive, with SERS detection of a few cells of Salmonella in neat samples achievable. We also evaluated the feasibility of using this device to detect Salmonella in food samples without performing the culturing procedures. Having spiked a few Salmonella cells into ice cubes and lettuce leaves, we use filtration and ultracentrifugation steps to obtain enriched tag-bound Salmonella samples of 200 µL. After loading an aliquot of 100 µL of sample onto this concentration device, the SERS tag signals from samples of 100 g ice cubes containing two Salmonella cells and 20 g lettuce leaf containing 5 Salmonella cells can be successfully detected.

Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis.

We developed a label-free, real-time, and highly sensitive nucleic acid biosensor based on fiber optic particle plasmon resonance (FOPPR). The biosensor employs a single-strand deoxyoligonucleotides (ssDNA) probe, conjugated to immobilized gold nanoparticles on the core surface of an optical fiber. We explore the steric effects on hybridization affinity and limit of detection (LOD), by using different ssDNA probe designs and surface chemistries, including diluent molecules of different lengths in mixed self-assembled monolayers, ssDNA probes of different oligonucleotide lengths, ssDNA probes in different orientations to accommodate target oligonucleotides with a hybridization region located unevenly in the strand. Based on the optimized ssDNA probe design and surface chemistry, we achieved LOD at sub-nM level, which makes detection of target oligonucleotides as low as 1 fmol possible in the 10-mL sensor chip. Additionally, the FOPPR biosensor shows a good correlation in determining HLA-B27 mRNA, in extracted blood samples from patients with ankylosing spondylitis (AS), with the clinically accepted real-time reverse transcription-polymerase chain reaction (RT-PCR) method. The results from this fundamental study should guide the design of ssDNA probe for anti-sense sensing. Further results through application to HLA-B27 mRNA detection illustrate the feasibility in detecting various nucleic acids of chemical and biological relevance.

Functional Biointerfaces Based on Mixed Zwitterionic Self-Assembled Monolayers for Biosensing Applications.

Surface modification for biosensors has focused attention for improvement of their sensitivity and specificity, particularly for the detection in complex medium. In this work, we have synthesized zwitterionic carboxybetaine-thiols (CB-thiols) and sulfobetaine-thiols (SB-thiols) for modification of gold substrates to form a functional self-assembled monolayer (SAM) for the immunoassay in a surface plasmon resonance (SPR) biosensor. X-ray photoelectron spectroscopy (XPS), contact angle goniometer, and cyclic voltammetry were applied for characterizations of elemental composition, surface wettability, and packing density, respectively. The antifouling properties of the SAMs were accessed by quantitative analysis of protein and bacterial adsorption. The results from the SAMs with a single component indicated that the SB-thiol SAM provides better surface hydrophilicity, fouling resistance, and packing density as compared to the CB-thiol SAM, likely due to the ionic association of CB moieties. However, the CB-thiol with the functional carboxylate group plays a critical role in postmodification of biomolecules via commercially available amine coupling chemistry. Thus, the mixed SAMs were prepared to integrate the unique characteristics from CB- and SB-thiols to control compositions and surface properties. The immunoassay was performed in the SPR biosensor, showing that the zwitterionic mixed SAM enables immobilization of biorecognition elements (BREs), and improved sensitivity and specificity. Consequently, the work reveals excellent and attractive versatility, antifouling, and functionalizable properties of zwitterionic mixed SAMs comprising CB- and SB-thiols for biosensing applications. This surface chemistry is expected to be applicable to monitor specific molecular recognition events.

Optofluidic refractive-index sensors employing bent waveguide structures for low-cost, rapid chemical and biomedical sensing.

We propose and develop an intensity-detection-based refractive-index (RI) sensor for low-cost, rapid RI sensing. The sensor is composed of a polymer bent ridge waveguide (BRWG) structure on a low-cost glass substrate and is integrated with a microfluidic channel. Different-RI solutions flowing through the BRWG sensing region induce output optical power variations caused by optical bend losses, enabling simple and real-time RI detection. Additionally, the sensors are fabricated using rapid and cost-effective vacuum-less processes, attaining the low cost and high throughput required for mass production. A good RI solution of 5.31 10-4 × RIU-1 is achieved from the RI experiments. This study demonstrates mass-producible and compact RI sensors for rapid and sensitive chemical analysis and biomedical sensing.

Effect of Surface Coverage of Gold Nanoparticles on the Refractive Index Sensitivity in Fiber-Optic Nanoplasmonic Sensing.

A simple theoretical model was developed to analyze the extinction spectrum of gold nanoparticles (AuNPs) on the fiber core and glass surfaces in order to aid the determination of the surface coverage and surface distribution of the AuNPs on the fiber core surface for sensitivity optimization of the fiber optic particle plasmon resonance (FOPPR) sensor. The extinction spectrum of AuNPs comprises of the interband absorption of AuNPs, non-interacting plasmon resonance (PR) band due to isolated AuNPs, and coupled PR band of interacting AuNPs. When the surface coverage is smaller than 12.2%, the plasmon coupling effect can almost be ignored. This method is also applied to understand the refractive index sensitivity of the FOPPR sensor with respect to the non-interacting PR band and the coupled PR band. In terms of wavelength sensitivity at a surface coverage of 18.6%, the refractive index sensitivity of the coupled PR band (205.5 nm/RIU) is greater than that of the non-interacting PR band (349.1 nm/RIU). In terms of extinction sensitivity, refractive index sensitivity of the coupled PR band (-3.86/RIU) is similar to that of the non-interacting PR band (-3.93/RIU). Both maximum wavelength and extinction sensitivities were found at a surface coverage of 15.2%.

Enhanced sensitivity in injection-molded guided-mode-resonance sensors via low-index cavity layers.

We present an investigation on the use of low-index cavity layers to enhance the sensitivity of injection-molded guided-mode resonance (GMR) sensors. By adjusting the sputtering parameters, a low-index cavity layer is created at the interface between the waveguide layer and the substrate. Refractive index measurements show that a sensitivity enhancement of up to 220% is achieved with a cavity layer, in comparison to a reference GMR sensor without a cavity layer. Finite-element-method simulations were performed, and the results indicate that the cavities significantly redistribute the resonance mode profile and thus enhances the sensitivity. The present investigation demonstrates a new method for enhancing the sensitivity of injection-molded GMR sensors for high-sensitivity label-free biosensing.

On-line SERS detection of single bacterium using novel SERS nanoprobes and a microfluidic dielectrophoresis device.

The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB-DEP-Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL(-1) . In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.

Noninvasive Prenatal Genetic Screening of Cell-Free Fetal DNA for Early Prediction of ß-Thalassemia Using Fiber Optic Nanogold-Linked Sorbent Assay.

ß-Thalassemia is a prevalent type of severe inherited chronic anemia, primarily identified in developing countries. The identification of single nucleotide polymorphisms (SNPs) plays a vital role in the early diagnosis of genetic diseases. Here, we reported the development of an amplification-free fiber optic nanogold-linked sorbent assay method using a fiber optic particle plasmon resonance (FOPPR) biosensor for rapid and ultrasensitive detection of SNPs. Herein, MutS protein was selected as the biorecognition capture probe and immobilized on the sensing region to capture the target mutant DNA, which was hybridized with a single-base mismatched single-stranded DNA labeled by a gold nanoparticle (AuNP). The AuNP acts as a signaling agent to be detected by the FOPPR biosensor when it is bound on the fiber core surface. The method effectively differentiates mismatched double-stranded DNA by MutS protein from perfectly matched/complementary dsDNA. It exhibits an impressively low detection limit for the detection of SNPs at approximately 10-16 M using low-cost sensor chips and devices. By determination of the ratio of mutant DNA to normal DNA in cell-free genomic DNA from blood samples, this method is promising for diagnosing ß-thalassemia in fetuses without invasive testing techniques.

Ultrasensitive Upconversion Nanoparticle Immunoassay for Human Serum Cardiac Troponin I Detection Achieved with Resonant Waveguide Grating.

Selective detection of biomarkers at low concentrations in blood is crucial for the clinical diagnosis of many diseases but remains challenging. In this work, we aimed to develop an ultrasensitive immunoassay that can detect biomarkers in serum with an attomolar limit of detection (LOD). We proposed a sandwich-type heterogeneous immunosensor in a 3 × 3 well array format by integrating a resonant waveguide grating (RWG) substrate with upconversion nanoparticles (UCNPs). UCNPs were used to label a target biomarker captured by capture antibody molecules immobilized on the surface of the RWG substrate, and the RWG substrate was used to enhance the upconversion luminescence (UCL) of UCNPs through excitation resonance. The LOD of the immunosensor was greatly reduced due to the increased UCL of UCNPs and the reduction of nonspecific adsorption of detection antibody-conjugated UCNPs on the RWG substrate surface by coating the RWG substrate surface with a carboxymethyl dextran layer. The immunosensor exhibited an extremely low LOD [0.24 fg/mL (9.1 aM)] and wide detection range (1 fg/mL to 100 pg/mL) in the detection of cardiac troponin I (cTnI). The cTnI concentrations in human serum samples collected at different times during cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) chemotherapy in a breast cancer patient were measured by an immunosensor, and the results showed that the CEF chemotherapy did cause cardiotoxicity in the patient. Having a higher number of wells in such an array-based biosensor, the sensor can be developed as a high-throughput diagnostic tool for clinically important biomarkers.