Vertical flow assays based on core-shell SERS nanotags for multiplex prostate cancer biomarker detection.

Rapid, simultaneous, and sensitive quantification of multiplex prostate biomarkers plays an important role in early diagnosis, especially for obese men and patients. Herein, a surface-enhanced Raman scattering (SERS)-based vertical flow assay (VFA) is presented for simultaneous detection of multiplex prostate cancer biomarkers, such as prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) on a single test spot. In practice, Raman dyes (RDs) encoded core-shell SERS nanotags instead of conventional gold colloids used in the colorimetric assay are employed in the sensing membrane of SERS based VFAs for multiplex protein detection. Because of the enhanced Raman signal of the core-shell nanostructure and the high surface area to volume ratio (SVR) of the porous sensing membrane, this proposed biosensor shows a wide linear dynamic range (LDR) with detection limits of 0.37, 0.43, and 0.26 pg mL-1 for PSA, CEA, and AFP, respectively, suggesting that this approach can be a good candidate in point of care testing (POCT) for rapid and sensitive biomarker detection and has a huge potential in multiplex analysis and cancer diagnosis.

A vertical flow microarray chip based on SERS nanotags for rapid and ultrasensitive quantification of α-fetoprotein and carcinoembryonic antigen.

A vertical flow microarray chip is described that uses core-shell SERS nanotags as tags for ultrasensitive quantification of the tumor markers α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) by detecting the intensity of the specific Raman bands at 592 cm-1. The nanotags warrant high sensitivity, and the use of porous nitrocellulose warrants a high surface-to-volume ratio. The linear dynamic ranges are 0.1 ng mL-1 - 10 µg mL-1 for both AFP and CEA, and the limits of detection) are 0.27 pg mL-1 and 0.96 pg mL-1, respectively. Quantification is rapid and can be performed without preconcentration. Graphical abstract Schematic representation of a vertical flow microarray chip using AuNBA@Ag SERS nanotags (where NBA stands for Nile blue A) as labels for rapid, ultrasensitive, and simultaneous detection of tumor biomarkers CEA and AFP.

Photonic Crystal Hydrogel Enhanced Plasmonic Staining for Multiplexed Protein Analysis.

Plasmonic nanoparticles are commonly used as optical transducers in sensing applications. The optical signals resulting from the interaction of analytes and plamsonic nanoparticles are influenced by surrounding physical structures where the nanoparticles are located. This paper proposes inverse opal photonic crystal hydrogel as 3D structure to improve Raman signals from plasmonic staining. By hybridization of the plasmonic nanoparticles and photonic crystal, surface-enhanced Raman spectroscopy (SERS) analysis of multiplexed protein is realized. It benefits the Raman analysis by providing high-density "hot spots" in 3D and extra enhancement of local electromagnetic field at the band edge of PhC with periodic refractive index distribution. The strong interaction of light and the hybrid 3D nanostructure offers new insights into plasmonic nanoparticle applications and biosensor design.

Transcriptomic profiling of nuclei from paraformaldehyde-fixed and formalin-fixed paraffin-embedded brain tissues.

BACKGROUND: Paraformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens. Single-cell gene expression workflows have recently been developed for PFA-fixed and FFPE tissue specimens. However, for tissues where intact cells are hard to recover, including tissues containing highly interconnected neurons, single-nuclear transcriptomics is beneficial. Moreover, since RNA is very unstable, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens like FFPE samples are largely anecdotal. RESULTS: We evaluated the effects of polyformaldehyde (PFA) fixation and paraffin-embedding on transcriptional profiles of the mouse hippocampus obtained by RNA sequencing (RNA-seq). The transcriptomic signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were comparable to those of cryopreserved samples. However, more differentially expressed genes were obtained for brains after PFA fixation for more than 3 days than in fresh PFA-fixed samples, especially genes involved in spliceosome and synaptic-related pathways. Importantly, the real cell states were destroyed, with oligodendrocyte precursor cells depleted in the 1day fixed hippocampus. After fixation for 3 days, the proportions of neuronal cells and oligodendrocytes decreased and microglia increased; however, relative frequencies remained constant for longer fixation durations. The storage time of FFPE samples had a negligible effect on the cell composition. SIGNIFICANCE: This represents the first work to investigate the effects of fixation and storage time of brains on its nuclear transcriptome signatures in detail. The fixation time had more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1-3 days. These findings are expected to guide sample preparation for single-nucleus RNA-seq of FFPE samples, particularly in transcriptomic studies focused on brain diseases.

Rapid and sensitive biomolecular screening with encoded macroporous hydrogel photonic beads.

We present a new method to prepare inverse opaline photonic beads with good spherical shape and superior optical performance by simply introducing an interfacial tension system into a template replication method. When the scaffolds of these beads were composed of poly(ethylene glycol) diacrylate hydrogel, they could provide a homogeneous water surrounding, which remedied many shortcomings of biomolecular microcarriers introduced by the presence of the solid surface of them. The suspension array, which used these macroporous hydrogel photonic beads as coding elements, showed obvious advantages in multiplexed capability, rapid biomolecular screening (within 12 min), and highly sensitive detection (with limit of detection of approximately 10(-12) M).

Colloidal crystal beads composed of core-shell particles for multiplex bioassay.

A convenient method was developed to fabricate colloidal crystal beads (CCBs) with tough mechanical strength, which was used as encoded carriers for multiplex bioassay. The latex particles used for the construction of the CCBs were designed with a rigid core PS and a elastomeric shell poly(MMA/EA/MAA), and were prepared via one-step soap-free emulsion polymerization. The as-above-prepared CCBs were thermo-treated to drive the elastomeric shells of adjacent latex particles joining together. It was found that the coalescence of latex particles can greatly improve the mechanical strength of the CCBs for multiplex bioassay.

Rapid and Ultrasensitive Quantification of Multiplex Respiratory Tract Infection Pathogen via Lateral Flow Microarray based on SERS Nanotags.

Respiratory tract infections (RTIs) are severe acute infectious diseases, which require the timely and accurate identification of the pathogens involved so that the individual treatment plan can be selected, including optimized use of antibiotics. However, high throughput and ultrasensitive quantification of multiple nucleic acids is a challenge in a point of care testing (POCT) device. Methods: Herein, we developed a 2×3 microarray on a lateral flow strip with surface enhanced Raman scattering (SERS) nanotags encoding the nucleic acids of 11 common RTI pathogens. On account of the signal magnification of encoded SERS nanotags in addition to the high surface area to volume ratio of the nitrocellulose (NC) membrane, rapid quantification of the 11 pathogens with a broad linear dynamic range (LDR) and ultra-high sensitivity was achieved on one lateral flow microarray. Results: The limit of detection (LOD) for influenza A, parainfluenza 1, parainfluenza 3, respiratory syncytial virus, coxiella burnetii, legionella pneumophila, influenza B, parainfluenza 2, adenovirus, chlamydophila pneumoniae, and mycoplasma pneumoniae were calculated to be 0.031 pM, 0.030 pM, 0.038 pM, 0.038 pM, 0.040 pM, 0.039 pM, 0.035 pM, 0.032 pM, 0.040 pM, 0.039 pM, and 0.041 pM, respectively. The LDR of measurement of the target nucleic acids of the eleven RTI pathogens were 1 pM-50 nM, which span 5 orders of magnitude. Conclusions: We anticipate this novel approach could be widely adopted in the early and precise diagnosis of RTI and other diseases.

Low cost 3D microfluidic chips for multiplex protein detection based on photonic crystal beads.

Point-of-care testing (POCT) devices used in multiplex bioassays are in great demand for clinical, environmental and biomedical applications. Photonic crystal beads (PCBs), as structural color self-coding carriers, can be integrated with microfluidic chips to realize convenient and highly sensitive biomarker detection. Here we developed a three dimensional (3D) microfluidic chip based on PCBs, which is low cost and easy to manufacture for mass production and application. The chip was fabricated with polyethylene terephthalate, polymethyl methacrylate sheets, a Ni square mesh grid and transparent double-sided tape. In practice, the target molecules could be captured by PCBs immobilized with probes in a flow-through manner. It was found that the as-proposed chip needed less washing and its background was effectively reduced in comparison with a flow-over chip. Besides, the limit of detection (LOD) of anti-human alpha fetoprotein (AFP) was calculated to be 18.92 ng mL-1, which could meet the need of clinical detection of AFP. Furthermore, the chip demonstrated the feasibility of simultaneous detection of human immunoglobulin G, carcinoembryonic antigen and AFP, which suggests that it has a broad application prospect in multiplex bioassays.

Fabrication of photonic crystals with nigrosine-doped poly(MMA-co-DVB-co-MAA) particles.

A convenient approach was developed to fabricate monodisperse nigrosine-doped poly(methyl methacrylate-co-divinylbenzene-co-methacrylic acid) nanoparticles with different cross-linkage by soap-free emulsion polymerization at boiling status and swelling process. The dye-doped nanoparticles were used for the fabrication of colloidal crystal films and beads. It was found that nigrosine dye in the nanoparticles can efficiently depress the light scattering inside the colloidal crystal films and eliminate the iridescent effect in the photonic beads. These results make the colloidal crystals useful in photonic paper, bioassay, and so on.

Fabrication of photo-encoded beads for bioanalysis.

Encoding large libraries of biomolecules is important to the applications such as immunoassay, drug screening and so on. By now a number of encoding schemes have been proposed and developed. In this paper, a new encoding approach was proposed based on the photonic crystal beads and an apparatus was developed for the fabrication. Encoded beads, with diameters in the range between 0.2 mm and 3 mm, were prepared using polymer and pearl dye. Pearl dye is a kind of photonic crystal, the color of which is from the ordered nanostructure. Such a colorizing mechanism makes the color of pearl dye much stable to high temperature, UV irradiation or longtime storage comparing with the conventional dyes.

Colorimetric photonic hydrogel aptasensor for the screening of heavy metal ions.

We have developed a robust method for the visual detection of heavy metal ions (such as Hg(2+) and Pb(2+)) by using aptamer-functionalized colloidal photonic crystal hydrogel (CPCH) films. The CPCHs were derived from a colloidal crystal array of monodisperse silica nanoparticles, which were polymerized within the polyacrylamide hydrogel. The heavy metal ion-responsive aptamers were then cross-linked in the hydrogel network. During detection, the specific binding of heavy metal ions and cross-linked single-stranded aptamers in the hydrogel network caused the hydrogel to shrink, which was detected as a corresponding blue shift in the Bragg diffraction peak position of the CPCHs. The shift value could be used to estimate, quantitatively, the amount of the target ion. It was demonstrated that our CPCH aptasensor could screen a wide concentration range of heavy metal ions with high selectivity and reversibility. In addition, these aptasensors could be rehydrated from dried gels for storage and aptamer protection. It is anticipated that our technology may also be used in the screening of a broad range of metal ions in food, drugs and the environment.

Photo-bleaching immunity encoded photonic suspension array for label-free multiplex analysis.

A new type of suspension array for multiplex analysis has been proposed by using polarized luminescent ratios of dichroic dyes as the encoding elements, which provided highly stable codes and advantages of label-free detection.