Advancements in Noble Metal Nanoparticles-Based Point-of-Care Testing: History
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Contributor:
Keven Luciano
,
Xiaochuan Wang
,
Yaning Liu
,
Gabriella Eyler
,
Zhenpeng Qin
,
Xiaohu Xia

Noble metal nanoparticles (NM NPs) have been used for POC testing for decades. The most known example might be the lateral flow assay (LFA, or test strip), where Au NPs are usually utilized as colorimetric labels owing to their outstanding optical properties. Over-the-counter pregnancy tests and the recent COVID-19 antigen rapid tests are representative examples of the lateral flow assays (LFA). Over the last couple of decades, engineered NM NPs have been extensively used for the point-of-care (POC) tests of various platforms beyond the LFA, despite most of them being in early stages of commercialization. This recent NM NPs-based POC testing techniques with innovative designs are discussed.

  • POC
  • NM NPs
  • plasmonic activities

1. Catalytically Active Noble Metal Nanoparticles-Based Point-of-Care Tests

Among noble metal nanoparticles (NM NPs), platinum-group metal (including Pt, Pd, Rh, Ir, and Ru) NPs are known to be excellent catalysts for many industrially important reactions. In recent years, these catalytic NM NPs have been employed to catalyze reactions that produce detectable signal for point-of-care (POC) testing.
In a recent work by Xia et al., conventional Au NPs of ~40 nm in diameter were coated with a thin layer of Pt to form Au@Pt core@shell NPs [1]. The Au@Pt NPs were able to effectively catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB, a typical peroxidase substrate) by H2O2, producing a blue-colored product oxidized TMB with a large molar extinction coefficient of 3.9 × 104 M−1 cm−1 [2][3]. The catalytic reaction can be conveniently performed in aqueous solution at room temperature, making it suitable for POC testing. Significantly, the color signal from Au@Pt NPs-catalyzed reaction is much stronger than the color signal from plasmonics of Au NPs, allowing for highly sensitive colorimetric detection. The Au@Pt NPs as labels were applied to the LFA platform. Using human prostate-specific antigen (PSA, a biomarker of prostate cancer) as a model disease biomarker, the Au@Pt NPs-based LFA achieved a low “naked eye” detection limit of 20 pg/mL, which was two orders of magnitude lower than that of conventional Au NPs-based LFA. In another work, Stevens et al. utilized porous Pt NPs to catalyze the oxidation of CN/DAB (4-chloro-1-naphthol/3,3′-diaminobenzidine, tetrahydrochloride) by H2O2 that generates black-colored products. The Pt NPs were applied to the LFA of p24 (a biomarker of HIV), achieving a low detection limit at the low femtomolar range [4]. Notably, this LFA system was successfully applied to the analyses of clinical human plasma samples.
NM NPs can also be utilized to catalyze reactions that generate signals other than color. Yang et al. reported an innovative POC testing system for circulating tumor cell (CTC) detection that was designed based on the oxygen gas generated by Pt NPs [5]. Specifically, in this system, target CTCs were captured and labeled with aptamer-conjugated Pt NPs. The Pt NPs can effectively catalyze the decomposition of H2O2, producing oxygen gas (O2). A portable volumetric bar chart chip (V-Chip) was coupled to the detection system. In the presence of target CTCs, the produced O2(g) results in movement of an ink bar in the V-Chip. As a result, the number of CTCs in a sample could be conveniently quantified by recording the distance moved by the ink. Such a portable POCT system was sensitive enough for single cell detection. In another design, O2(g) generated by NM NPs (e.g., Pt NPs and Au@AgPt NPs) was retained in a confined space [6]. An increased amount of O2(g) led to an increase in gas pressure that could be read by a portable pressuremeter. As such, the concentration of target analytes could be quantitively determined by measuring the gas pressure.

2. Plasmonically Active Noble Metal Nanoparticles-Based Point-of-Care Tests

Plasmonic NM NPs (e.g., Au and Ag NPs) have found wide applications in POC tests [7]. Bimetallic nanostructures, such as gold-silver nanocages, have attracted significant research interest due to the tunable LSPR properties [8][9]. Particularly, their refractive index sensitivity can be effectively regulated by the wall thickness and ratio of Au to Ag. Conventional Au-Ag cages prepared by the galvanic replacement between Ag NPs as templates and HAuCl4 are confined to a specific wall thickness [10]. Gao et al. adopted a template regeneration strategy in galvanic replacement reaction to craft the Au-Ag nanocages with controllable wall thicknesses and intriguing plasmonic properties [11]. Particularly, the wall of nanocages can be controlled to the desired thickness using regenerated templates (i.e., Ag@Au-Ag core@shell nanostructures) for continuous galvanic replacement. With the well-defined multiwall morphologies and the disappearance of the surface cavities, the LSPR of newly developed Au-Ag nanocages shifted from 775 nm to the visible range of 551 nm. To demonstrate the potential application in POC testing, [Ag-Au]5 nanocages (i.e., nanocages of five-layered walls) with λmax of ~550 nm (red color) were applied as labels to the LFA to detect the human prostate-specific antigen (PSA). The results suggested that [Ag-Au]5 nanocages achieved a naked eye detection limit at 0.1 ng mL−1, which was ~10 times lower than that of conventional Au NP-based LFA.
Plasmonic coupling assays (PCAs) are another class of rapid tests for a broad range of analytes from proteins to virus particles. The LSPR of NM NPs shifts when NPs come in close proximity to each other (e.g., aggregations) and gives an observable color change. Since the initial report by Mirkin et al. in 1997 [12], NM NPs-based PCAs have been extensively employed in various sensing applications, including the sample-to-answer detection of aptamers, proteins, viruses, and bacteria, in diverse biologically complex media to diagnose infectious diseases [13]. Previous work has demonstrated that the plasmonic properties of MN NPs have strong dependence on various parameters, such as their size, morphologies, the composition of metal, and the surrounding environments. Recently, Ye et al. developed a simpler method for preparing Au-Ag nanoshells with enhanced plasmonic activities [14]. Rather than repeating the galvanic replacement reaction on the regenerated templates, they performed the reaction in the presence of Na3CA. Upon injecting the HAuCl4, the Na3CA quickly reduced the Au3+ ions into Au+, such that the stoichiometry between Au and Ag in the galvanic replacement reaction changed from 1:3 to 1:1. The resulting Au-Ag nanoshells with hollow interiors show superior plasmonic activities due to the field enhancement from the plasmon hybridization between the inner and outer surfaces. The Energy-dispersive X-ray (EDX) mapping image of an individual Au-Ag nanoshell confirmed the elemental distribution, where Au and Ag elements are diffused throughout the NPs. Compared with the same size solid Au NPs (50 nm) at the same particle concentration, Au-Ag nanoshells have four times higher extinction cross-section at visible wavelength range and 20-fold improvement in detecting DNA. When integrating with reverse transcription loop-mediated isothermal amplification (RT-LAMP), Au-Ag nanoshells realized the single-molecule detection of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) RNA with high specificity. Liu et al. further demonstrated that altering nanoparticle morphology has a significant importance on the intact virion detection [15]. With respiratory syncytial virus, they demonstrated that Au nanourchins have increased capability to bind to the virus particle compared with spherical Au NP, and stronger plasmonic coupling at longer distances (~10 nm) that are relevant for immunorecognition.

3. Photothermally Active Noble Metal Nanoparticles-Based Point-of-Care Tests

The absorption of light energy by NM NPs leads to photothermal heating and can serve as sensitive contrast. Qin et al. first reported a thermal contrast amplification (TCA) strategy for Au NP-based LFAs with continuous wave laser heating [16]. By applying laser on a completed LFA strip, the accumulated Au NPs on the test line induce temperature changes that can be directly recorded by an infrared camera or sensor. Compared with visual detection, TCA readout provides improved ability in the analytical quantification of LFA results  [17]. Later optimization of the immunoassays and miniaturization of the TCA instrumentations by Zhan et al. further enhanced the LFA sensitivity up to 256-fold  [17]. Notably, the design of NM NPs as thermal contrast labels has a significant impact on the LFA reaction kinetics and TCA signal, thus affecting the LFA analytical performance. For example, the larger Au NPs hold higher binding affinity to the target analyte due to more antibody conjugation on the Au NP and increased Au NP capture. Combined with the high light absorption and scattering for larger Au NP, they allow much more sensitive detection. Other factors, such as the low diffusion limit for large NPs and highly non-specific background signals caused by membrane-trapping, should also be considered.
While the continuous wave (CW) laser heating leads to a bulk temperature increase, pulsed laser can excite the NM NPs locally and vaporize water to create nanobubbles, referred to as plasmonic nanobubbles (PNBs). Liu et al. utilized the digital PNB (dPNB) detection for intact virus diagnosis [18]. Since the vapor and liquid water have very different refractive indexes, dPNB can be easily detected by a continuous laser probe.  An optofluidic setup was designed to flow the Au NP suspensions in a micro-capillary for high throughput detection. The focused laser beams create a microscale “virtual detection zone” of about 16 pL and detect dPNB signals. There is no crosstalk between laser pulses since PNB only last hundreds of nanoseconds. This allows for the rapid counting of dPNBs and set thresholds for “on” and “off” signals in a compartment-free manner. When implemented in a homogeneous assay for respiratory syncytial viruses (RSV) detection, dPNB achieved a limit of detection at ~100 PFU/mL or 1 genome-equivalent copy/µ. This is competitive with nucleic acid amplification methods. Further advantages include the simplicity of the assay without separation or amplification steps, room temperature operation, and rapid dPNB counting, within minutes. Such a system opens new possibilities to develop separation-, amplification-, and compartment-free NM NP-based digital assay that is a rapid and ultrasensitive POC diagnostic platform.

4. Surface-Enhanced Raman Scattering Active Noble Metal Nanoparticles-Based Point-of-Care Tests

The Raman signal of molecules can be drastically enhanced by metallic nanoparticles (particularly Ag and Au NPs) owing to the localized electromagnetic field around the surface of NPs [19]. This phenomenon is known as surface-enhanced Raman scattering (SERS), whereas the NPs are called SERS substrates [20]. Since the pioneer work by Van Duyne et al. in 1977, SERS has been broadly used for biosensing applications [21][22]. The recent development of portable or handheld Raman spectrometer makes SERS suitable for POC testing.
In the 2000s and early 2010s, great effort in the field of SERS biosensors had been put on engineering sensitive SERS substrates with large enhancement factors (EFs). In particular, EF of a substrate can be substantially increased through the formation of hot spots (i.e., small, localized regions with intensified electric fields [23]). Common methods for the fabrication of hot spots include engineering nanostructures with sharp features (e.g., corners and edges) and inducing nanoparticle aggregations [24].
In recent years, the trend of fabricating hybrid SERS substrate has drawn increasing attention, where NM NPs are incorporated with secondary functional materials [25]. Hybrid SERS substrates can integrate the merits of multiple materials and/or produce synergies. For instance, by coupling NM NPs with semiconductors, SERS EF can be enhanced by ~10–103-fold through combined (synergistic) contributions from both materials. In a typical hybrid noble metal-semiconductor system, photoexcited electrons arising from the LSPR of metal flow to conduction band of semiconductor. Such a process promotes a semiconductor-to-molecule charge transfer process, resulting in a chemical mechanism-based SERS enhancement [25]. This synergistic enhancement had been demonstrated in the Au-TiO2 system [26]. In another example, noble metal was coupled with carbon nanotubes [27]. Specifically, single-walled carbon nanotubes (SWCNTs) were functionalized with Ag/Au alloyed NPs to form SWCNT/Ag/AuNPs conjugates. The 2D-band of SWCNTs at 2578 cm–1 remains unchanged and thus can be used as the internal reference. This hybrid SERS substrate allows for more reliable and reproducible detection because the signal is measured by ratiometric intensity between SWCNT as an internal reference and a Raman reporter molecule (e.g., MPP with a peak at 2207 cm−1).
Another important progress of SERS active NM NPs-based POC testing is to address emerging healthcare issues. A notable strategy is to use SERS tags (i.e., SERS active NPs pre-functionalized with reporter molecules with known Raman peaks) as labels for the LFA. As a distinct advantage over conventional LFAs, SERS tag-based LFA is more sensitive because a small amount of SERS tags specifically captured in the test line of LFA strip can provide strong Raman signal. In a recent study by Wang et al., Raman dye-functionalized SiO2@Ag core@shell NPs were used as SERS tags for LFA of anti-SARS-CoV-2 (the virus that causes COVID-19) IgM and IgG [28]. The SERS signal intensities of the IgM and IgG test lines were conveniently recorded by a portable Raman instrument. The detection limit of this SERS tag-based LFA was 800 times lower than that of standard Au NPs-based LFA. Significantly, the SERS tag-based LFA was successfully applied to serum samples collected from COVID-19 patients, demonstrating the potential clinical use of the new technology. The platform of SERS tag-based LFA can also be applied to detection of other infectious diseases. For instance, Choo et al. developed a SERS LFA for serodiagnosis of scrub typhus, a mite-borne infectious disease [29].

5. Label-Free Colorimetric Noble Metal Nanoparticles-Based Point-of-Care Tests

Owing to the outstanding optical properties, NM NPs (especially Au and Ag) have been demonstrated to be excellent colorimetric labels for POC testing where the detection results can be visualized by naked eyes. Importantly, the color of Au and Ag NPs can be tuned in the visible light spectrum by controlling NP morphology (e.g., size and shape) and/or elemental composition [30][31], which allows for the design of innovative POC tests, such as those capable of multiplexed detection.
In recent years, label-free colorimetric NM NPs have been utilized for the development of versatile and sensitive POC tests [32]. In this system, colorimetric NM NPs are not labeled with bioreceptors, which reduces the non-specific binding of NPs caused by bioreceptors and improves detection reproducibility. In a typical design, target analytes in an assay are linked to the generation of certain substance that can trigger the morphological or compositional changes of colorimetric NPs through creative mechanisms (e.g., growth and etching of NPs).
In a recent work by Xia et al., Au/Ag alloyed nanocages are used as label-free colorimetric reporters for the detection of human carcinoembryonic antigen (CEA, a cancer biomarker) [33]. In this detection system, CEA is specifically captured by antibodies that are labeled with alkaline phosphatase (ALP). ALP can effectively catalyze the formation of ascorbic acid that induces the growth of Ag on the inner surfaces of Au/Ag nanocages. As the amount of Ag inside the nanocages is increased (which is correlated to CEA concentration), a distinct color change from light blue to blue, violet, magenta, and orange, can be visualized. As such, the concentration of CEA in a sample can be conveniently determined by comparing the color of assay solution with the color chart of CEA standards of known concentrations. It should be noted that, compared to the growth of Ag on the surface of solid NPs (e.g., Au nanospheres and nanorods), the growth of Ag inside Au/Ag nanocages is more efficient in tuning the color of NP suspension. This advantage ensures a high detection sensitivity of the Au/Ag nanocages-based detection platform.
In another work by Yang et al., the color change of NP suspension was achieved through chemical etching [34]. Specifically, target antigen HIV-1 p24 was specifically captured by horseradish peroxidase (HRP)-labeled antibodies. HRP-catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) can quantitatively mediate the etching of Au nanorods (Au NRs). The aspect ratio (length/width) of Au NRs was reduced as the extent of etching was increased, which led to various color changes. The assay was performed in a microfluidic platform that enables the integration of all analytical processing within one small chip, making the detection technique particularly suitable for POC testing.

6. Noble Metal Nanoparticles-Based Point-of-Care Tests of Other Mechanisms

In addition to the above mentioned systems, POC tests can be designed and established by taking advantage of other properties of NM NPs through various mechanisms. For example, the average hydrodynamic size of NM NPs can be measured by dynamic light scattering (DLS). The measured size is highly sensitive to the change in the refractive index of surrounding medium of NPs and the coupling or aggregation of NPs [35]. Therefore, NM NPs can be employed for the development of DLS-based POC biosensors. NM NPs are also used in electrochemical biosensors that rely on amperometry or voltammetry techniques [36]. In this approach, NM NPs can enhance electrochemical signal through various mechanisms, such as increasing the loading of electrochemically detectable species and catalyzing the electrolysis of a large amount of substrate [37]. NM NPs of ultra-small sizes (<2 nm), possess fluorescent properties, allowing for the development of fluorescent biosensors [38][39]. In some recent studies, NM NPs were used for developing biosensors with creative mechanisms. For instance, Au nanorods are responsive to the acoustic field, which can induce particle aggregation [40]. Such induced aggregation can be integrated with Raman enhancement for sensitive and rapid biosensing.

This entry is adapted from the peer-reviewed paper 10.3390/bioengineering9110666

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