•A novel dose-dependent multicolor conversion strategy is developed for colorimetric bioassays.•AuNP-mediated copper deposition is employed for signal amplification.•Semi-quantitative PSA detection was demonstrated with naked eyes.•The sensing platform can be easily adapted to detection of other biomolecules.Colorimetric immunoassays have been attracting more attention for use in practical applications, especially in point−of−care diagnostics. In comparison with a single color immunoassay, the dose−dependent multicolor strategy greatly improves the detection resolution and accuracy of visual inspection. In the current study, a concentration−dependent multicolor conversion strategy was developed based on gold nanoparticle (AuNP)−mediated copper deposition for signal amplification and Prussian blue for color generation. Under optimal conditions, a dose−dependent multicolor from yellow through green to blue were successfully achieved, which was easier to be differentiated from each other by the naked eyes. With rabbit IgG and prostate specific antigen (PSA) as model analytes, semi−quantitative evaluations were demonstrated in lab buffer and serum by direct readout with the naked eyes. Quantitative detections were also accomplished by measurement of absorbance of Prussian blue with a common UV–Vis spectrophotometer. A limit of detection (LOD) down to sub−picogram per milliliter was determined. In addition, this newly developed colorimetric assay method can be easily adapted for the detection of other biomolecules by simply changing the recognition pairs.Download high-res image (181KB)Download full-size image

•An ultrasensitive colorimetric immunoassay is developed based on iodine–starch system.•Glucose oxidase (GOx) efficiently catalyzes cascade signal amplification.•Qualitative detection can be realized through direct readout with naked eyes.•High sensitive PSA detection was successfully demonstrated in serum.•The sensing system could be easily adapted to detection of other biomolecules.This work reports a novel colorimetric immunoassay for ultrasensitive quantitation of prostate specific antigen (PSA) based on glucose oxidase (GOx) catalyzed cascade formation of blue-black iodine–starch complex. In this design, a potassium iodide (KI)–starch system was integrated with a sandwich immunoassay, multivalent biotin–avidin interaction and high efficient GOx catalysis. In the presence of PSA, biotinylated anti–PSA and GOx–conjugated avidin was successively introduced via the specific immunobinding and the high affinity biotin–avidin interaction. GOx–catalyzed oxidation of glucose leads to cascade production of gluconic acid and hydrogen peroxide (H2O2), which subsequently oxidizes KI and results in a visual color change of the starch solution due to the formation of an intense colored iodine–starch complex. The color intensity is proportional to the analyte level, allowing a simple and rapid assay by direct readout with naked eyes. A quantitative immunoassay is easily accomplished by an UV–vis spectrophotometer. Under optimal condition, a colorimetric immunoassay with an ultralow limit of detection (LOD) at 0.46 pg mL−1 and a wide dynamic range from 1 pg mL−1 to 1 μg mL−1 was achieved with PSA as a model biomarker, demonstrating its great potential for early cancer diagnosis in clinical application.A novel colorimetric immunoassay for ultrasensitive quantification of prostate specific antigen (PSA) based on glucose oxidase (GOD) catalyzed cascade formation of blue-black iodine–starch complexation. An ultralow detection limit at 0.46 pg mL−1 and a wide dynamic range from 1 pg mL−1 to 1 μg mL−1 was achieved with PSA as a model biomarker, demonstrating its great potential for early cancer diagnosis in clinical application.Download high-res image (89KB)Download full-size image

Although bioenzymes or artificial nanoenzymes-catalyzed reactions can efficiently amplify the detection signal in immunosorbent assays, the cascade reactions are sensitive to various conditions, including pH, temperature, time, and so on, resulting in poor reproducibility and dose-dependent response. In this work, a novel colorimetric immunoassay strategy was developed using an iron oxide magnetic nanoparticle (MNP) as a label for signal generation and amplification without the need for subsequent cascade reactions. The signal intensity is directly proportional to the level of targeting analytes, providing a more accurate quantification and a better reproducibility. The measurable color change results from the formation of a soluble red complex between bathophenanthrolinedisulfonic acid disodium salt (BPT) and Fe2+ released from dissolution of iron oxide MNP. The MNP, acting as an iron pool, provides a large amount of Fe2+ after its dissolution, thus promoting an efficient signal amplification. A qualitative evaluation is directly observed with the naked eye, while quantitative detection is achieved by measuring the absorbance of the colored solution using a UV-Vis spectrophotometer. Under optimal conditions, a limit of detection (LOD) at 3.6 pg mL−1 was achieved with a cancer biomarker carcinoembryonic antigen (CEA) as a model analyte, demonstrating its great potential for early cancer diagnosis. Moreover, the developed colorimetric assay method can be easily adapted to detection of other biomolecules (nucleic acids, saccharides, small molecules and so on) by simply changing the recognition pairs.

Immobilization of proteins on a solid support is critical with respect to the fabrication and performance of biosensors and biochips. Protein attachment with a preferable orientation can effectively avoid its denaturation and keeps its active sites fully exposed to solution, thus maximally preserving the bioaffinity or bioactivity. This review (with 140 refs.) summarises the recent advances in oriented immobilization of proteins with a particular focus on antibodies and enzymes. Following an introduction that describes reasons for oriented immobilization on (nano)surfaces, we summarize (a) methods for (bio)chemical affinity-mediated oriented immobilization (with sections on immunoglobulin G (IgG)-binding protein as the capture ligand, DNA-directed immobilization, aptamer- and peptide-mediated immobilization, affinity ligand and fusion tag-mediated immobilization, material-binding peptide-assisted immobilization); (b) methods for covalent oriented immobilization (with sections on immobilization via cysteine residues or cysteine tags, via carbohydrate moieties; via enzyme fusion or enzymatic catalysis, and via nucleotide binding sites of antibodies); (c) methods based on molecular imprinting techniques; (d) methods for characterization of oriented immobilized proteins; and then make conclusions and give perspectives.

•A new conceptual 3-D array fluidic immunoassay device is developed.•The device is made by simply coupling a glass cuboid into a circular tube without microfabrication process.•The 3-D cuboid substrate offers fourfold effective surface for more sensing spots.•The device greatly enhances the mass transport for rapid immunoassay.Conventional 2-D microarray is known to have high-throughput detection capability; however, the sensing spots density is significantly hindered by the spot-to-spot distance (gap) requirement for eliminating cross-talks between adjacent spots. Herein a new conceptual 3-D microarray device is proposed to significantly improve the spots density. To demonstrate advantages of the 3-D array, a microfabrication-free fluidic immunoassay device is further made by simply coupling an antibodies-arrayed glass cuboid into a circular glass tube. Rapid, sensitive and high-throughput flow-through immunoassays were accomplished with the 3-D array-based device for detection limits of 10–100 pg mL−1 and wide dynamic range over 4–5 orders of magnitude in human serum with cancer biomarkers α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model targets, holding great promise for practical clinical applications. The 3-D microarray device not only significantly increases the density of sensing spots, but also greatly enhances the mass transport for rapid immunoassay when using in a flow-through device.

•Polymer brush with boronic acid is prepared on glass slide.•High density and oriented antibody immobilization is achieved on boronic acid brush.•Oriented immobilized antibody renders high bioactivity and signal-to-noise ratio.•Highly sensitive antibody microarray is demonstrated by sandwich immunoassay.There still has a big challenge for low-abundance protein detection with antibody microarrays. In this work, phenylboronic acid (PBA) polymer brush-enabled oriented, high density, and covalent antibody immobilization was realized for sensitive antibody microarrays. PBA-enabled oriented antibody attachment via carbohydrate at Fc portion keeps antigen-binding sites fully expose to their corresponding antigens, resulting in higher antibody–antigen (Ab–Ag) binding efficiency. Sandwich immunoassay with rabbit IgG as model analyte was performed on poly(glycidyl methacrylate)-amino-phenylboronic acid-coated glass slide (PGMA-APBA-slide). One order improvement of LOD was achieved as compared with that on poly(glycidyl methacrylate) glass slide (PGMA-slide). The improvement is mainly attributed to PBA-assisted high density and oriented antibody immobilization. This work provides a versatile and effective strategy to develop high sensitive antibody microarrays for low-abundance protein analysis in various proteomic applications.

Although bioenzymes or artificial nanoenzymes-catalyzed reactions can efficiently amplify the detection signal in immunosorbent assays, the cascade reactions are sensitive to various conditions, including pH, temperature, time, and so on, resulting in poor reproducibility and dose-dependent response. In this work, a novel colorimetric immunoassay strategy was developed using an iron oxide magnetic nanoparticle (MNP) as a label for signal generation and amplification without the need for subsequent cascade reactions. The signal intensity is directly proportional to the level of targeting analytes, providing a more accurate quantification and a better reproducibility. The measurable color change results from the formation of a soluble red complex between bathophenanthrolinedisulfonic acid disodium salt (BPT) and Fe2+ released from dissolution of iron oxide MNP. The MNP, acting as an iron pool, provides a large amount of Fe2+ after its dissolution, thus promoting an efficient signal amplification. A qualitative evaluation is directly observed with the naked eye, while quantitative detection is achieved by measuring the absorbance of the colored solution using a UV-Vis spectrophotometer. Under optimal conditions, a limit of detection (LOD) at 3.6 pg mL−1 was achieved with a cancer biomarker carcinoembryonic antigen (CEA) as a model analyte, demonstrating its great potential for early cancer diagnosis. Moreover, the developed colorimetric assay method can be easily adapted to detection of other biomolecules (nucleic acids, saccharides, small molecules and so on) by simply changing the recognition pairs.