Title：M13 Bacteriophage-Assisted Recognition and Signal Spatiotemporal Separation Enabling Ultrasensitive Light Scattering Immunoassay
Journal: ACS Nano
IF: 17.1
Original link: https://pubs.acs.org/doi/full/10.1021/acsnano.3c07194
Reporter: Xiatong Wang, Master of Grade 2022
The sensitivity of traditional immunoassay-based detection methods is limited due to the contradiction between molecular recognition and signal amplification caused by the size effect of nanoprobes. This study utilized a multifunctional M13 phage assisted immune recognition and signal transduction spatiotemporal separation, and a highly sensitive light scattering immunoassay system was used to quantitatively detect low abundance target analytes. Given the synergistic effect of M13 phage mediated leverage and GISG amplified light scattering signal modulation, the actual detection capability of this strategy can achieve ultra sensitive and rapid quantification of ochratoxin A and alpha fetoprotein in real samples at sub femolar levels within 50 minutes. In order to further improve the sensitivity of our immunoassay, a biotin streptavidin amplification protocol was implemented to detect the 2-spike protein of severe acute respiratory syndrome coronavirus, with sensitivity reaching the Amor range. In summary, this study provides direction for the ultra sensitive quantitative detection of target analytes through the synergistic combination of M13 phage mediated leverage effect and GISG amplified light scattering signal modulation.
Immunoassay plays a crucial role in the measurement of low abundance target analytes due to its sensitivity, specificity, and accurate quantitative detection. Typically, immunoassay methods use specific antigen antibody recognition to manipulate analyte concentration dependent signal transduction for quantitative detection of target analytes in samples. With the rapid development of signal transduction, immunoassay technology has gradually evolved from traditional colorimetric assays to detection based on fluorescence, chemiluminescence, plasma, electrochemistry, Raman scattering, and mass spectrometry. Given these significant advancements, the analytical sensitivity of immunoassays has been significantly improved, with detection limits comparable to PCR. Despite these achievements, innovation in new methods and technologies for ultra sensitive target detection is still urgent and highly challenging.
Efficient molecular recognition and highly sensitive signal transduction are key to achieving high-performance immunoassay. Compared with traditional single signal molecule labeling, using micro/nano carriers carrying a large number of signal molecules as signal amplification probes can theoretically improve sensitivity, as large-sized micro/nano carriers contain more signal molecules than small-sized carriers. However, due to the slow diffusion rate of signal probes from the bulk solution to the target capture surface, as well as the steric hindrance effect caused by the increase in nanoprobe size, the immune response efficiency at the sensing element/transducer interface is low. Therefore, most surface analysis techniques have limitations in terms of reaction kinetics and sensitivity.
This work developed a universal ultra sensitive identification and signal spatiotemporal separation light scattering immunoassay method for quantitative detection of low abundance target analytes. Specifically, the M13 bacteriophage selectively recognizes target analytes at the pIII protein through phage display technology using nanoantibodies, and further chemically modifies with biotin molecules to increase the interference of small AuNPs modified with streptavidin (SA) on the M13 bacteriophage. Magnetic nanoparticles (MNPs) coated with antibodies or antigens are used to guide the assembly of genetically engineered M13 bacteriophages through specific immune recognition.
1.Amplified Consumption of AuNP Seeds by M13 Bacteriophage-Mediated Leverage Effect
Figure 1. (a) Schematic illustration of the binding of small-sized AuNPs to M13 bacteriophages based on the streptavidin–biotin recognition. (b) The laser scanning confocal microscopy imaging for verifying the successful conjugation of biotin molecule with M13 bacteriophage. The average (c) hydrodynamic diameter and (d) UV–visible absorption spectra of AuNPs, AuNP@SA, M13 + AuNP@SA, and M13@biotin + AuNP@SA. (e) TEM image of MNP@mAb + M13OTA@biotin + AuNP@SA.
2. M13 Phage-Driven Competitive Light Scattering Immunoassay for Small Molecules
Figure 2. (a) Schematic illustration of the principle of GISG strategy for enlarging the AuNP size. (b) TEM images, (c) hydrodynamic diameter distribution, and (d) UV–vis absorption spectra of AuNP@SA before and after GISG treatment. (e) Light scattering intensity changes and (f) absorbance changes at 512 and 561 nm of AuNPs at different AuNP@SA concentrations before and after GISG treatment.
3. M13 Phage-Driven Competitive Light Scattering Immunoassay for Small Molecules
Figure 3. (a) Schematic illustration of the workflow of M13OTA@biotin-driven competitive light scattering immunoassay. (b) Competitive inhibition rates against concentrations of a series of OTA standard solutions ranging from 0.01 fg mL–1 to 16 pg mL–1. (c) Quantitative calibration curve of the established competitive light scattering immunoassay method for OTA quantitation. (d) Specificity evaluation for OTA (10 pg mL–1) detection of this immunoassay to other common interfering mycotoxins including ZEN, AFB1, AFB2, DON, and CIT with the concentration of 10 ng mL–1. (e) Correlation analysis between the detection results obtained from the proposed light scattering immunoassay and the HPLC method in detecting OTA corn samples. (f) Morphology images of MNP@mAb-M13OTA@biotin-AuNP@SA under different OTA concentrations (1, 30, 300, 3000 fg mL–1) observed by TEM.
4.M13 Phage-Driven Sandwich Light Scattering Immunoassay for Biomacromolecules
Figure 4. (a) Schematic illustration of the workflow of M13AFP@biotin-driven sandwich light scattering immunoassay. (b) Light scattering intensity against concentrations of a series of AFP standard solutions ranging from 0 pg mL–1 to 20 ng mL–1. (c) Quantitative calibration curve of AFP using the established sandwich light scattering immunoassay method. (d) Specificity evaluation for AFP (20 ng mL–1) determination of this immunoassay to other common interfering disease protein biomarkers including PCT, CEA, CRP, PSA, and HBsAg with the concentration of 20 ng mL–1. (e) Correlation analysis between the test results obtained from the proposed light scattering immunoassay and the commercial chemiluminescence immunoassay kits in quantifying AFP serum samples.
5. Performance Verification of M13 Phage-Driven Light Scattering Immunoassay in Pandemic Management
Figure 5. (a) Schematic illustration of the working principle of M13Nb@biotin-driven competitive light scattering immunoassay for SARS-CoV-2 spike (S) protein. (b) Schematic illustration of biotin–streptavidin amplification scheme for measuring SARS-CoV-2 S protein. Competitive inhibition rates against concentrations of a series of S protein standard solutions ranging from 0 pg mL–1 to 10 pg mL–1 (c) without and (e) with the biotin–streptavidin amplification scheme. Quantitative calibration curve of S protein using the developed competitive light scattering immunoassay (d) without and (f) with the biotin–streptavidin amplification scheme. (g) TEM image showed more M13 phages around each MNP. (h) High-resolution image further confirmed many small-sized AuNP@SA were attached to the filamentous surface of M13 bacteriophage.
(1) This study establishes a multifunctional M13 phage assisted recognition and signal spatiotemporal separation strategy to achieve an ultrasensitive, fast, and broad-spectrum light scattering immunoassay system.
(2) This method achieved ultrasensitive and rapid quantification of trace analytes at the subfemtomolar level within 50 minutes, and compared with traditional phage ELISA, the detection sensitivity increased by about 4 orders of magnitude.
(3) By integrating the biotin-SA amplification scheme, the sensitivity of SARS-CoV-2S protein was further improved to the Amor range.