Xiangwei Zhao

Encoded Porous Beads for Label‐Free Multiplex Detection of Tumor Markers

In recent years, suspension arrays, which use self-encoded microcarriers as elements, are attracting increasing interest in the field of drug discovery, gene-function analysis, clinical diagnosis, and so on. Compared with the conventional microarrays on a plate, suspension arrays may offer greater flexibility in the preparation of new assays, higher diffusional flux of analytes due to the radial diffusion, less consumption of sample and reagents, and higher sensitivity. One of the key techniques of suspension arrays is encoding. Spectrum is a wellused encoding approach, due to its simplicity both in encoding and decoding. Fluorescent dyes and quantum dots are the main spectrum-encoding elements, and the beads encoded by fluorescence have been commercialized by Luminex and other companies. However, fluorescence dyes tend to be quenched or bleached, and the quantum dots are usually biotoxic. Also, the fluorescence of the carriers can interfere with the signal from the labeling molecules, and as a result affect the detection limit. Photonic crystals have been suggested as a new type of spectrum-encoding carrier, whose code is the characteristic reflection peak originated from the stop-band. As the peak position is based on their periodical structure, the code is very stable, and the fluorescent background is low. These properties render photonic crystals suitable for highly sensitive detection. Conventional planar photonic-crystal carriers have to be properly dispersed and correctly orientated to avoid stacking or standing of the flakes during the decoding process. Recently, we found that these problems might be solved with the use of photonic beads with a 3D structure. Unlike planar photonic-crystal carriers, the photonic beads retain their reflection spectra when the crystals are rotated under light impinging on the crystal surface at a fixed incident angle. This indicates the accurate encoding character of photonic beads. Another key technique for using suspension arrays is target analysis, which is generally realized by detecting the labels attached to the biomolecules. In addition to the high cost imposed by reagents and instruments, the labels, or labeling itself, will hinder the interactions and the activity of biomolecules. Therefore, label-free detection is anticipated. In this paper, we proposed the use of inverse-opaline photonic beads as carriers for suspension arrays. The beads showed large encoding capacity by changing their lattice constant. A label-free multiplex detection of tumor markers, Human CA125, CA19-9, and CEA, which showed great significance in early screening and clinical diagnosis of some tumor diseases including colorectal cancer, gastric cancer, and lung cancer, show the flexibility and feasibility of our suspension array in clinical applications. In addition, both the decoding and bioreaction detection were measurements of the characteristic reflection peak, which rendered the detection and the analyzing apparatus extremely simple. Up to now, attention was devoted to the fabrication of inverseopaline photonic films, and only a few reports on the formation of inverse-opaline photonic beads are available. Template replication and spray-drying with colloidal templates are examples of methods that showed the difficulty in preparing beads with smooth edges and long-range-ordering porous surfaces, due to low surface tension and too fast assembly. Herein, we produced inverse-opaline photonic beads by colloidal crystallization in droplet templates. First, an aqueous suspension containing monodisperse polystyrene spheres and ultrafine silica particles was broken into droplets by oil flows in a microfluidic device, and the droplets were taken into a collection container that was also filled with silicon oil. Then, the polystyrene spheres self-assembled into ordered lattices, and the ultrafine silica particles infiltrated into the interstitial sites between the spheres during the evaporation of water in the droplets. After solidification, the hybrid beads were thoroughly washed with hexane to remove silicon oil. Finally, the hybrid beads were calcined, to remove the polystyrene spheres and improve the mechanical strength of the inverseopaline photonic beads. Generally, the long-range ordering of pores on bead surfaces was important for the optical performance of the inverse-opaline photonic beads. To optimize this performance, change of pore arrangement on the bead surface with varying polystyrene spheres to silica particles volume ratios in the droplet template were investigated. Theoretically, when the polystyrene spheres packed closely to a bead in a face-centered cubic arrangement, and the ultrafine silica particles infiltrated all the interstitial sites between the polystyrene spheres, the volume ratio was about 3.85. In our experiment, however, we found that during the evaporation of water the polystyrene spheres escaped more easily from the droplet templates than silica particles; also, after solidification the silica particles could not infiltrate into all the interstitial sites

Encoded Silica Colloidal Crystal Beads as Supports for Potential Multiplex Immunoassay

We developed a new kind of suspension array for multiplexed immunoassays using silica colloidal crystal beads (SCCBs) as coding carriers. The monodisperse and size-controlled SCCBs were fabricated by a microfluidic device. Calcination was employed to improve the mechanical stability and lower the fluorescent background of the SCCBs. Immobilization of protein molecules on the surface of the SCCBs through chemical bonds was studied, and the modification condition was optimized to increase the detection sensitivity. Results indicated that the SCCBs as supports were more sensitive (0.92 ng/mL IgG) than the glass beads (27 ng/mL IgG) and the planar carriers (140 ng/mL IgG). A multiplex immunoassay showed the flexibility and feasibility of SCCBs array in clinical applications.

Multiplex Label-Free Detection of Biomolecules with an Imprinted Suspension Array†

Multiplex detection of biomolecules has facilitated clinical diagnosis, drug discovery, and environmental testing. Immunoassays using labeled antibodies are the most popular methods for the detection of biomolecules because of their sensitivity and selectivity. However, labeling protocols are time-consuming and expensive, and may alter the form or physicochemical behavior of the antibodies, which could lead to false negative results. In addition, the reagent stability, high cost, and difficulties associated with antibody production, together with the adverse action of toxic compounds or immunosuppressants on the metabolism and the immune system during the production of antibodies, are often cited as problems. Therefore, it is highly desirable to carry out multiplex label-free detection of biomolecules without using immunoassay methods. Suspension arrays, which use self-encoded microcarriers as elements, have shown obvious advantages in the multiplex detection of biomolecules. However, few of them can be used for the label-free detection of biomolecules. Moreover, these methods are still based on the use of probe antibodies. In contrast, molecularly imprinted polymers (MIPs) have unique properties as mimics of natural molecular receptors that may make them suitable for revolutionary applications in biotechnology. Recently, many kinds of MIP sensors have been developed for the detection of biomolecules based on physicochemical responses of the MIPs, such as changes in refractive index and volume. However, these sensors could respond only to single analytes, and the detection signals from the physicochemical response of the MIPs were not distinct and were difficult to measure with accuracy. Herein, we report a new type of suspension array for the multiplex label-free detection of biomolecules without using immunoassay methods. The microcarriers of our suspension array are molecularly imprinted polymer beads (MIPBs) with photonic crystal structure, which not only provide diffraction peaks for encoding but also convert the slight physicochemical response signals to the obvious changes of optical signals. This technique combines the advantages of suspension arrays, molecular imprinting, and photonic crystal sensors. As a proof of concept for the multiplex label-free detection of biomolecules not based on immunoassay methods, we constructed an imprinted suspension array with affinity for proteins. For large biomacromolecules, surface imprinting was used for the fabrication of the MIPBs with photonic crystal structure due to the easy removal and rapid rebinding characteristics during assays. Scheme 1 outlines the

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.

Multiplex detection of tumor markers with photonic suspension array

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL(-1), 1.0-500 ng mL(-1), 1.0-500 U mL(-1) and 3.0-500 U mL(-1) with limits of detection of 0.68 ng mL(-1), 0.95 ng mL(-1), 0.99 U mL(-1) and 2.30 U mL(-1) at 3 sigma, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.

Photonic crystal hydrogel beads used for multiplex biomolecular detection

A microfluidic device was used to generate droplets of monodispersed silica nanoparticles in poly (ethylene glycol) diacrylates (PEG-DA), which were then polymerized into hydrogel beads by UV irradiation. The nanoparticles in the beads were assembled into ordered lattices and locked in the network of the hydrogel. Therefore, the beads showed photonic crystal features, whose reflection peaks were used as the coding elements of biomolecular carriers. And huge coding capacity could be gained by changing the volume fraction of the silica nanoparticles in the droplets. The beads provided homogeneous water surroundings so that some shortcomings in bioassay introduced by the presence of solid surface of carriers could be avoided. Their application in oligonucleotide detection demonstrated potential advantages of hydrogel beads as encoded carriers in multiplex biomolecular detection.

Fabrication of Colloidal Crystal Beads by a Drop‐Breaking Technique and Their Application as Bioassays

Multiplex assay is a critical technique for drug discovery, combinatorial chemistry, and diagnostics. A number of reactions occur simultaneously and each of them should be tracked in the course of the assay. One approach for multiplex assay is the use of planar-array chips, on which a large number of molecular probes can be immobilized. Each of them is in a definite position and encoded by its coordination. Although this method has the advantage of large encoding capacity, and is easy for encoding and decoding, the reaction speed is diffusion-limited, which can make detection slow. Besides, this approach has problems in its inflexibility of use and spotting variance. An alternative approach is bonding the molecular probes onto encoded supports that can be mixed with the analytes, which are known as fluidic microcarriers. In this method, uniquely encoded microcarriers immobilized with molecular probes can be mixed together in a floating system and subjected to an assay. One critical technique for using fluidic microcarriers is microcarrier encoding. To date, a number of approaches have been proposed to encode the microcarriers, among which optical encoding based on fluorescence from dyes or quantum dots has been extensively studied. Most of the commercialized products are based on this approach. However, the organic dyes or quantum dots have the disadvantages of photobleaching and chemical instability during storage. These problems become serious when accompanied by an increase of code capacity. Recently, photonic crystals have appeared as a new type of encoding approach that can solve these problems. The photonic crystal is a kind of material with an ordered structure. It reflects light whose wavelength falls into its stop-band region. Sailor et al. proposed the use of periodically etched porous silicon slices as the microcarriers. One disadvantage is that the reflection peak which is used for encoding shifts with the detection angle, as the periodical structure is one-dimensional, and carriers should be well positioned during detection to obtain the correct code. In

Three-dimensionally Ordered Macroporous （3DOM） Gold-nanoparticle Doped Titanium Dioxide （GTD） Photonic Crystals Modified Electrodes for Hydrogen Peroxide Biosensor

Gold nanoparticles have been introduced into the wall framework of titanium dioxide photonic crystals by the colloidal crystal template technique. The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface and used for the hydrogen peroxide biosensor. The direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) immobilized on this film have been investigated. The 3DOM GTD film could provide a good microenvironment for retaining the biological bioactivity, large internal area, and superior conductivity. The HRP/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the HRP intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.19 and -0.52V in 0.1M PBS (pH 7.4), respectively. The HRP intercalated in the mesopores showed a surface-controlled process with a single proton transfer. The direct electron transfer between the adsorbed HRP and the electrode is achieved without the aid of an electron mediator. The H(2)O(2) biosensor displayed a rapid eletrocatalytic response (less than 3s), a wide linear range from 0.5 microM to 1.4mM with a detection limit of 0.2 microM, high sensitivity (179.9 microAmM(-1)), good stability and reproducibility. Compared with the free-Au doped titanium dioxide photonic crystals modified electrode, the GTD modified electrode could greatly enhance the response current signal, linear detection range and higher sensitivity. The 3DOM GTD provided a new matrix for protein immobilization and direct transfer study and opened a way for low conductivity electrode biosensor.

Colloidal crystal beads coated with multicolor CdTe quantum dots: microcarriers for optical encoding and fluorescence enhancement

A novel type of microcarrier was developed for optical encoding and fluorescence enhancement by depositing semiconductor quantum dots (QDs) on silica colloidal crystals beads (SCCBs). Monodisperse SCCBs were used as the support to deposit different-sized and different-layer QDs, thus leading to wavelength-and-intensity coding. The unique properties of QDs were well suited for multiplexed optical encoding, which could potentially yield a large number of codes. The SCCBs possess high porosity and high surface-to-volume ratio (SVR), which can provide stronger detection signals and thus high detection sensitivity. DNA hybridization studies demonstrated that the detection limit for this kind of microcarrier (6.7 pmol/L) was much lower than that for glass beads (890 pmol/L). These results indicate that designed optical encoding microcarriers could be successfully applied to high-throughput and multiplexed biomolecular assays.

Macroporous ordered titanium dioxide (TiO2) inverse opal as a new label-free immunosensor

Photonic crystal sensing materials have been validated that they are very sensitive to refractive index changes. Herein, three-dimensionally ordered macroporous (3DOM) (>50 nm) TiO2 inverse opal film has been fabricated by the self-assembly technique. Based on the TiO2 inverse opal film, the optical spectrometer was established for label-free immunosensor. The sensing performance of the 3DOM TiO2 was investigated using human IgG/goat anti-human IgG couple, which showed that the sensitivity of 3DOM TiO2 inverse opal film could reach to 1 microg mL(-1) (equivalent to 1.5 pg mm(-2)) of protein concentration detection limit. The 3DOM TiO2 inverse opal has a large internal surface area, low fluorescence background and unique optical properties. These characteristics indicated the feasibility of 3DOM TiO2 inverse opal in label-free immunoassay.