Nanoparticle-based Immunoassays Research Articles

Advancing Diagnostics: Evaluating the Potential of Nanoparticle-Based Immunoassays as PCR Alternatives for Disease Confirmation

Abstract: In diagnostic technologies, searching for quick, accurate, and cost-efficient ways to confirm diseases has prompted the investigation of alternative methodologies to classic PCR-based tests. This article delves into the growing field of nanoparticle-based immunoassays, offering a comprehensive evaluation of their potential as viable alternatives to PCR for disease diagnostics. We discuss the basic principles of nanoparticle-based immunoassays, highlighting their distinctive capacity to combine the specialized binding characteristics of antibodies with the improved detection capacities of nanoparticles. The article compares the performance attributes of nanoparticle-based immunoassays compared to PCR, focusing on sensitivity, specificity, and detection speed. We further delve into a novel diagnostic technology, immuno-PCR, which integrates the strengths of immunoassays and PCR techniques. Healthcare systems and stakeholders must comprehend and implement novel diagnostic procedures emphasizing accuracy and sensitivity as the diagnostic landscape advances globally. This review contributes to the advancement of diagnostics by synthesizing current research and suggesting future directions for development. It highlights the transformative potential of current and emerging methods in shaping the future of disease diagnostics.

Comparison of single and double pulse laser-induced breakdown spectroscopy for the detection of biomolecules tagged with photon-upconversion nanoparticles

BackgroundLaser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity. ResultsThis paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL−1 to 0.29 ng mL−1. In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples. Significance and noveltyIn this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique.

Facemask analyses for the non-invasive detection of chronic and acute P. aeruginosa lung infections using nanoparticle-based immunoassays.

Pseudomonas aeruginosa (P. aeruginosa) is a pathogen that persistently colonizes the respiratory tract of patients with chronic lung diseases. The risk of acquiring a chronic P. aeruginosa infection can be minimized by rapidly detecting the pathogen in the patient's airways and promptly administrating adequate antibiotics. However, the rapid detection of P. aeruginosa in the lungs involves the analysis of sputum, which is a highly complex matrix that is not always available. Here, we propose an alternative diagnosis based on analyzing breath aerosols. In this approach, nanoparticle immunosensors identify bacteria adhered to the polypropylene layer of a surgical facemask that was previously worn by the patient. A polypropylene processing protocol was optimized to ensure the efficient capture and analysis of the target pathogen. The proposed analytical platform has a theoretical limit of detection of 105 CFU mL-1 in aerosolized mock samples, and a dynamic range between 105 and 108 CFU mL-1. When tested with facemasks worn by patients, the biosensors were able to detect chronic and acute P. aeruginosa lung infections, and to differentiate them from respiratory infections caused by other pathogens. The results shown here pave the way to diagnose Pseudomonas infections at the bedside, as well as to identify the progress from chronic to acute infection.

The application of nanoparticles in point-of-care testing (POCT) immunoassays.

The Covid-19 pandemic has led to greater recognition of the importance of the fast and timely detection of pathogens. Recent advances in point-of-care testing (POCT) technology have shown promising results for rapid diagnosis. Immunoassays are among the most extensive POCT assays, in which specific labels are used to indicate and amplify the immune signal. Nanoparticles (NPs) are above the rest because of their versatile properties. Much work has been devoted to NPs to find more efficient immunoassays. Herein, we comprehensively describe NP-based immunoassays with a focus on particle species and their specific applications. This review describes immunoassays along with key concepts surrounding their preparation and bioconjugation to show their defining role in immunosensors. The specific mechanisms, microfluidic immunoassays, electrochemical immunoassays (ELCAs), immunochromatographic assays (ICAs), enzyme-linked immunosorbent assays (ELISA), and microarrays are covered herein. For each mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining the biosensing and related point-of-care (POC) utility. Given their maturity, some specific applications using different nanomaterials are discussed in more detail. Finally, we outline future challenges and perspectives to give a brief guideline for the development of appropriate platforms.

Fast and ultrafast thermal contrast amplification of gold nanoparticle-based immunoassays

For highly sensitive point-of-care (POC) diagnostics, we explored the limit of thermal contrast amplification (TCA) reading of gold nanoparticles (GNPs/mm2) at test regions in immunoassays. More specifically, we built and compared fast (minute scale) and ultrafast (seconds scale) TCA setups using continuous-wave (CW) and ms pulsed lasers, respectively. TCA improved the limit of detection (LoD) for silica-core gold nanoshells (GNSs) preloaded in nitrocellulose (NC) membrane as model lateral flow immunoassays (LFAs) by 10- to 20-fold over visual reading. While the ultrafast TCA led to higher thermal signals, this came with a twofold loss in LoD vs. fast TCA primarily due to noise within the infrared sensor and a necessity to limit power to avoid burning. To allow higher laser power, and therefore amplification fold, we also explored transparent glass coverslip substrate as a model microfluidic immunoassay (MIA). We found the ultrafast TCA reading of GNS-coated coverslips achieved a maximal signal amplification (57-fold) over visual reading of model LFAs. Therefore, ultrafast TCA-MIA is promising for ultrasensitive and ultrafast diagnostics. Further advantages of using TCA in MIA vs. LFA could include lower sample volume, multiplexed tests, higher throughput, and fast reading. In summary, TCA technology is able to enhance the sensitivity and speed of reading GNPs (GNPs/mm2) within both LFAs and MIAs.

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-based Immunoassays.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 µm in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Laser-induced breakdown spectroscopy as a novel readout method for nanoparticle-based immunoassays.

Laser-induced breakdown spectroscopy (LIBS) was examined as a novel method for readout of microtiter plate immunoassays involving nanoparticles (NP). The so-called Tag-LIBS technique is a sensitive method for the detection of specific biomarkers. It was applied to the determination of NP labels using nanosecond ablation sampling. The NP labels were examined from the bottom of a standard 96-well microtiter plate. Thanks to the flexibility of LIBS instrumentation, both the plasma emission collection and the focusing optics arrangements can be collinearly arranged. The experiments showed that silver NPs and gold NPs can be readily quantified on the bottom of the microtiter plate. Utilizing this technique, a sandwich immunoassay for human serum albumin using streptavidin-coated AgNP labels was developed. The assay has a 10ng·mL-1 detection limit which is comparable to the sensitivity of fluorometric readout. The main advantage of this LIBS technique is its wide scope in which it enables a detection of almost any type of NP labels, irrespective to any fluorescence or catalytic properties. Owing to the immediate signal response, the relatively simple instrumentation also enables assay automation. The LIBS capability of multi-elemental analyses makes it a promising and fast alternative to other readout techniques, in particular with respect to multiplexed detection of biomarkers. Graphical abstract Laser-induced breakdown spectroscopy (LIBS) is used as a novel readout method of nanoparticle-based immunoassays in microtiter plates. After formation of sandwich immunocomplex, the analyte concentration is quantified as the signal of Ag nanoparticle labels determined by LIBS.

Highly sensitive nanoparticle-based immunoassays with elemental detection: Application to Prostate-Specific Antigen quantification

One of the major challenges in developing novel assay methods for the detection of biomolecules is achieving high sensitivity, because of the ultralow concentrations typically in clinical samples. Here, a Mn-doped ZnS quantum dots-based immunoassay platform is presented for highly sensitive detection of cancer biomarkers. Ultrahigh sensitivity is achieved through gold deposition on the surface of the nanoparticle tags acting as catalytic seeds, thus effectively amplifying the size of the metallic nanoparticles after the immunoassay and before the tag detection. Elemental mass spectrometry measurement of the gold content allowed detection of Prostate-Specific Antigen (PSA) at the low attog mL−1 level. Moreover, the developed method showed not only an extremely high sensitivity for PSA detection but also a broad dynamic range, higher than 8 orders of magnitude, particularly useful for clinical studies involving quantitative detection of diverse biomarkers at their very different relevant concentration levels. Its applicability to discriminate small differences in PSA concentrations at low levels (few pgmL−1) in real serum samples was successfully evaluated.

A plasmonic nanosensor for immunoassay via enzyme-triggered click chemistry.

Current techniques for plasmonic immunoassay often require the introduction and additional conjugation of enzyme, and thus cannot accommodate conventional immunoassay platforms. Herein, we develop a plasmonic nanosensor that well accommodates conventional immunoassays and dramatically improves their sensitivity and stability. This plasmonic nanosensor directly employs alkaline phosphatase-triggered click chemistry between azide/alkyne functionalized gold nanoparticles as the readout. This straightforward approach broadens the applicability of nanoparticle-based immunoassays and has great potential for applications in resource-constrained settings.

Extension of dynamic range of sensitive nanoparticle-based immunoassays

Nanoparticles have successfully been employed in immunometric assays that require high sensitivity. Certain analytes, however, require dynamic ranges (DRs) around a predetermined cut-off value. Here, we have studied the effects that antibody orientation and addition of free solid-phase and detection antibodies have on assay sensitivity and DR in traditional sandwich-type immunoassays. D-dimer and cardiac troponin I (cTnI), both routinely used in critical care testing, were applied as model analytes. The assays were performed in microtitration wells with preimmobilized solid-phase antibody. Inherently fluorescent nanoparticles coated with second antibody were used to detect the analyte. The selection of antibody orientation and addition of free solid-phase or detection antibody, with nanoparticles and calibrator, desensitized the assays and extended the DR. With D-dimer the upper limit of the DR was improved from 50 to 10,000ng/ml, and with cTnI from 25 to 1000ng/ml. Regression analysis with the Stago STA Liatest D-dimer assay yielded a slope (95% confidence interval) of 0.09 (0.07–0.11) and a y-intercept of −7.79 (−17.87–2.29)ng/L (n=65, r=0.906). Thus it is concluded that Europium(III)-chelate-doped nanoparticles can also be employed in immunoassays that require wide DRs around a certain cut-off limit.

Nanoparticle-based immunoassays in the biomedical field

Recent research has looked to develop innovative, powerful and novel biofunctionalized nanoparticles, controlling and tailoring their properties in a very predictable manner to meet the needs of clinic immunoassays in the biomedical field. This minireview briefly summarizes recent advances covering the last 3 years, exploiting nanoparticle-based electrochemical, optical, mass-sensitive, colorimetric and immunodipstick assays. The enormous signal enhancement associated with the use of nanoparticles and formation of nanoparticle-antibody-antigen assemblies provide the basis for sensitive detection of disease-related proteins or biomarkers. Rather than being exhaustive, this minireview focuses on selected examples to illustrate novel concepts and promising applications. Finally, a small amount of speculation of possible future developments in nanoparticle-based immunoassays is provided.