Fluorescence-Based Lateral Flow Immunoassay for Quantification of Infliximab: Analytical and Clinical Performance Evaluation.

BACKGROUND: Therapeutic drug monitoring of infliximab (IFX) can improve treatment outcomes; however, the temporal gap between drug concentration monitoring and subsequent availability restricts its practical application. To address this issue, an automated monitoring method, AFIAS IFX, was developed to rapidly and accurately analyze IFX concentration in blood. The analytical and clinical performances of this method were assessed to establish its clinical utility. METHODS: The analytical performance of AFIAS IFX was evaluated according to Clinical and Laboratory Standard Institute guidelines. For clinical validation, AFIAS IFX was compared with 3 established enzyme-linked immunosorbent assay kits (LISA TRACKER, RIDASCREEN, and ImmunoGuide) using 100 consecutive samples from 28 patients treated with IFX. Passing-Bablok regression and Bland-Altman analyses were performed to compare the methods. RESULTS: The detection and quantification limits of AFIAS IFX were 0.12 and 0.20 mcg/mL, respectively. Furthermore, AFIAS IFX analyzed samples within 10 minutes for concentrations up to 50 mcg/mL, exhibiting reproducibility (coefficient of variation [CV] ≤7.8%) and accuracy (recovery 98%-101%) with serum, plasma, and whole blood samples. Clinically, it exhibited a good correlation with the 3 established enzyme-linked immunosorbent assay kits. For patients treated with Remicade (IFX), the Passing-Bablok regression slope was 1.001-1.259, with a mean difference of -1.48 to 0.28 mcg/mL. For patients treated with CT-P13, the Passing-Bablok regression slope was 0.974-1.254, with a mean difference of -2.44 to 0.15 mcg/mL. CONCLUSIONS: AFIAS IFX, a novel fluorescence-based lateral flow assay, exhibited excellent performance in analyzing IFX trough levels and is a potentially powerful tool for therapeutic drug monitoring in clinical settings, with opportunities for further development.

Highly Sensitive Detection of Hormone Estradiol E2 Using Surface-Enhanced Raman Scattering Based Immunoassays for the Clinical Diagnosis of Precocious Puberty.

The hormone estradiol (17ß-estradiol, E2) plays an important role in sexual development and serves as an important diagnostic biomarker of various clinical conditions. Particularly, the serum E2 concentration is very low (<10 pg/mL) in prepubertal girls. Accordingly, many efforts to develop a sensitive method of detection and quantification of E2 in human serum have been made. Nonetheless, current clinical detection methods are insufficient for accurate assessment of E2 at low concentrations (<10 pg/mL). Thus, there is an urgent need for new technologies with efficient and sensitive detection of E2 for use in routine clinical diagnostics. In this study, we introduce a new E2 assay technique using a surface-enhanced Raman scattering (SERS)-based detection method. The SERS-based assay was performed with 30 blood samples to assess its clinical feasibility, and the results were compared with data obtained using the ARCHITECT chemiluminescence immunoassay. Whereas the commercial assay system was unable to quantify serum levels of E2 lower than 10 pg/mL, the limit of detection of E2 using the novel SERS-based assay described in this study was 0.65 pg/mL. Thus, the proposed SERS-based assay has a strong potential to be a valuable tool in the early diagnosis of precocious puberty due to its excellent analytical sensitivity.

Clinical validation of surface-enhanced Raman scattering-based immunoassays in the early diagnosis of rheumatoid arthritis.

We assessed the clinical feasibility of conducting immunoassays based on surface-enhanced Raman scattering (SERS) in the early diagnosis of rheumatoid arthritis (RA). An autoantibody against citrullinated peptide (anti-CCP) was used as a biomarker, magnetic beads conjugated with CCP were used as substrates, and the SERS nanotags were comprised of anti-human IgG-conjugated hollow gold nanospheres (HGNs). We were able to determine the anti-CCP serum levels successfully by observing the distinctive Raman intensities corresponding to the SERS nanotags. At high concentrations of anti-CCP (>25 U/mL), the results obtained from the SERS assay confirmed those obtained via an ELISA-based assay. Nevertheless, quantitation via our SERS-based assay is significantly more accurate at low concentrations (<25 U/mL). In this study, we compared the results of an anti-CCP assay of 74 clinical blood samples obtained from the SERS-based assay to that of a commercial ELISA kit. The results of the anti-CCP-positive group (n = 31, >25 U/mL) revealed a good correlation between the ELISA and SERS-based assays. However, in the anti-CCP-negative group (n = 43, <25 U/mL), the SERS-based assay was shown to be more reproducible. Accordingly, we suggest that SERS-based assays are novel and potentially useful tools in the early diagnosis of RA.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment.

SERS-based competitive immunoassay of troponin I and CK-MB markers for early diagnosis of acute myocardial infarction.

We report a SERS-based competitive immunoassay technique for the early diagnosis of acute myocardial infarction (AMI). Simultaneous quantification of the dual cardiac markers, CK-MB and troponin I, was achieved by single wavelength excitation.

Fabrication of SERS-fluorescence dual modal nanoprobes and application to multiplex cancer cell imaging.

We report a highly sensitive optical imaging technology using surface-enhanced Raman scattering (SERS)-fluorescence dual modal nanoprobes (DMNPs). Fluorescence microscopy is a well-known imaging technique that shows specific protein distributions within cells. However, most currently available fluorescent organic dyes have relatively weak emission intensities and are rapidly photo-bleached. Thus more sensitive and stable probes are needed. In this work we develop DMNPs, which can be used for both SERS and fluorescence detection. SERS detection is a powerful technique that allows ultrasensitive chemical or biochemical analysis through unlimited multiplexing and single molecule sensitivity. Combining advantages of fluorescence and SERS allows these dual modal nanostructures to be used as powerful probes for novel biomedical imaging. In this work, the fabrication and characterization of the SERS-fluorescence DMNPs and application to biological imaging were investigated using markers CD24 and CD44, which are co-expressed in MDA-MB-231 breast cancer cells, as a model system. SERS imaging with DMNPs was found to be a powerful tool to determine the co-localization of CD24 and CD44 in the cell.

Simultaneous immunoassay for the detection of two lung cancer markers using functionalized SERS nanoprobes.

A quick and reproducible SERS-based immunoassay, using functionalized hollow gold nanospheres and magnetic beads, has been developed. Here, a simultaneous detection of dual cancer markers in blood serum has been achieved under a single excitation wavelength. The accuracy and sensitivity for clinical sera from five patients confirms their diagnostic feasibility.

Preparation of silica-encapsulated hollow gold nanosphere tags using layer-by-layer method for multiplex surface-enhanced raman scattering detection.

The use of silica shells offers many advantages in surface-enhanced Raman scattering (SERS)-based biological sensing applications due to their optical transparency, remarkable stability in environmental media, and improved biocompatibility. Here, we report a novel layer-by-layer method for the preparation of silica-hollow gold nanosphere (HGN) SERS tags. Poly(acrylic acid) was used to stabilize Raman reporter-tagged HGNs prior to the adsorption of a coupling agent, after which a silica shell was deposited onto the particle surface using Stöber's method. Importantly, competitive adsorption of the Raman reporter molecules and coupling agents, which results in unbalanced loading of reporter molecules on individual nanoparticles, was avoided using this method. As a result, the loading density of reporter molecules could be maximized. In addition, HGNs exhibited strong enhancement effects from the individual particles because of their ability to localize the surface electromagnetic fields through pinholes in the hollow particle structures. The proposed layer-by-layer silica-encapsulated HGN tags showed strong SERS signals as well as excellent multiplexing capabilities.

Optoelectrofluidic sandwich immunoassays for detection of human tumor marker using surface-enhanced Raman scattering.

A sandwich immunoassay is a powerful tool for identifying a specific substance in a biological sample. However, its heterogeneous strategy always requires repetitive liquid handlings and long processing time. Here an optoelectrofluidic immunoassay platform for simple, fast, and automated detection of human tumor marker based on surface-enhanced Raman scattering (SERS) has been developed. By using a conventional optoelectrofluidic device and a liquid crystal display module, simple and quantitative detection of human tumor marker, alpha-fetoprotein, in a ∼500 nL sample droplet has been automatically conducted with lower detection limit of about 0.1 ng/mL within 5 min. This study depicts the first practical application, for protein detection, of the optoelectrofluidic manipulation technology. This image-driven immunoassay platform opens a new way for simple, fast, automated, and highly sensitive detection of antigens.

On-chip immunoassay using surface-enhanced Raman scattering of hollow gold nanospheres.

A surface-enhanced Raman scattering (SERS)-based gradient optofluidic sensor has been developed for a fast and sensitive immunoassay. In this work, a novel microfluidic sensor with functional internal structures has been designed and fabricated. This sensor is composed of three compartments consisting of the gradient channel that serially dilutes the target marker, the injection and mixing area of antibody-conjugated hollow gold nanospheres and magnetic beads, and the trapping area of sandwich immunocomplexes using multiple solenoids. Quantitative analysis of a specific target marker is performed by analyzing its characteristic SERS signals. This SERS-based gradient optofluidic sensor can replace the set of microwells or microtubes used in manual serial dilutions that have been traditionally used in enzyme-linked immunosorbent assay (ELISA)-type assays. The limit of detection for rabbit immunoglobin (IgG) is estimated to be 1-10 ng/mL. This novel SERS-based optofluidic immunoassay system is expected to be a powerful clinical tool for the fast and sensitive medical diagnosis of a disease.

Surface-enhanced Raman scattering in nanoliter droplets: towards high-sensitivity detection of mercury (II) ions.

We report a new method for the trace analysis of mercury (II) ions in water. The approach involves the use of droplet-based microfluidics combined with surface-enhanced Raman scattering (SERS) detection. This novel combination provides both fast and sensitive detection of mercury (II) ions in water. Specifically, mercury (II) ion detection is performed by using the strong affinity between gold nanoparticles and mercury (II) ions. This interaction causes a change in the SERS signal of the reporter molecule rhodamine B that is a function of mercury (II) ion concentration. To allow both reproducible and quantitative analysis, aqueous samples are encapsulated within nanoliter-sized droplets. Manipulation of such droplets through winding microchannels affords rapid and efficient mixing of the contents. Additionally, memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, are removed since the aqueous droplets are completely isolated by a continuous oil phase. Quantitative analysis of mercury (II) ions was performed by calculating spectral peak area of rhodamine B at 1,647 cm(-1). Using this approach, the calculated concentration limit of detection was estimated to be between 100 and 500 ppt. Compared with fluorescence-based methods for the trace analysis of mercury (II) ions, the detection sensitivities were enhanced by approximately one order of magnitude. The proposed analytical method offers a rapid and reproducible trace detection capability for mercury (II) ions in water.

Highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced Raman scattering of hollow gold nanospheres.

A quick and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay technique, using hollow gold nanospheres (HGNs) and magnetic beads, has been developed. Here, HGNs show strong enhancement effects from individual particles because hot spots can be localized on the pinholes in the hollow particle structure. Thus, HGNs can be used for highly reproducible immunoanalysis of cancer markers. Magnetic beads were used as supporting substrates for the formation of the immunocomplex. This SERS-based immunoassay technique overcomes the problem of slow immunoreaction caused by the diffusion-limited kinetics on a solid substrate because all of the reactions occur in solution. For the validation of our SERS immunoassay, a well-known lung cancer marker, carcinoembryonic antigen (CEA), was used as a target marker. According to our experimental results, the limit of detection (LOD) was determined to be 1-10 pg/mL, this value being about 100-1000 times more sensitive than the LOD of enzyme-linked immunosorbent assay. Furthermore, the assay time took less than 1 h, including washing and optical detection steps.

Surface-enhanced Raman scattering imaging of HER2 cancer markers overexpressed in single MCF7 cells using antibody conjugated hollow gold nanospheres.

Antibody-conjugated hollow gold nanospheres (HGNs) have been used for the SERS imaging of HER2 cancer markers overexpressed in single MCF7 cells. SERS mapping images show that HGNs have much better homogeneous scattering properties than silver nanoparticles. The results demonstrate the potential feasibility of HGNs as highly sensitive and homogeneous sensing probes for biological imaging of cancer markers in live cells.