Table 3 Overview of nanoplasmonic biosensor studies for immune profiling

SARS-CoV-2 IgG, purified and in human plasma samples

Gold triangular nanoprisms

(~53 nm edge length)

Recombinant antigen consisting of SARS-CoV-2 spike 1 (S1) protein subunit or linear epitopes thereof, attached via crosslinker

-Antibody samples were incubated with antigen-functionalized surface overnight prior to aqueous rinsing and endpoint measurement.

-Measurement response showed linearity over ~11 orders of magnitude (10^-9 to 10²nM) for different analytes (up to ~12 nm shifts).

-Optimal ratio of two linear epitope probes achieved ~30 aM limit of detection (tenfold improvement).

-Selectively detected virus-specific antibodies and screened over 100 plasma samples from COVID-19 patients and controls (90% specificity and 100% sensitivity).

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SARS-CoV-2 IgG, purified and in immunized mouse sera; influenza A virus IgG, purified

Gold nanospikes

(~50 nm diameter)

Recombinant antigen consisting of SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) and full-length influenza hemagglutinin (HA) 1 and 2, which were biotinylated and attached to a streptavidin coating

-Antibody samples were incubated with antigen-functionalized surface for 2 h prior to aqueous rinsing and air-drying, followed by endpoint measurement.

-RBD antigen-functionalized sensor detected 10 ng/mL monoclonal SARS-CoV-2 IgG with ~4 nm shift and was insensitive to influenza A virus IgG (~0.3 nm shift).

-HA1- and HA2-functionalized sensors detected 10 ng/mL influenza IgG with ~2–4 nm shift and was insensitive to SARS-CoV-2 IgG (<0.3 nm shift).

-Detected polyclonal SARS-CoV-2 IgG in mouse sera after immunization with full-length SARS-CoV-2 spike protein (0.1 nm before vs. 2.7 nm shifts after immunization).

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SARS-CoV-2 IgG, purified and in human serum samples

Gold nanoholes

(~200 nm diameter and ~500 nm periodicity)

Secondary antibody (anti-IgG) to sensor chip and recombinant antigen consisting of SARS-CoV-2 S protein RBD to nanoparticle amplifier, both attached via noncovalent adsorption

-Antibody samples and antigen-functionalized nanoparticle signal amplifiers were sequentially added and then co-incubated with secondary antibody-functionalized surface for 15 min prior to endpoint optical density (OD) measurement.

-Two types of nanoparticle amplifiers were tested, with measured antibody detection ranges of 2–400 pM (nanoporous hollow gold) and 34–2176 pM (solid colloidal gold).

-Hollow nanoparticles achieved an estimated limit of detection of 0.2 pM and had a nonlinear, sigmoidal concentration-response curve.

-Selectively detected SARS-CoV-2 IgG antibodies in 139 serum samples from vaccinated individuals (96% specificity and 89% sensitivity).

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SARS-CoV-2 neutralizing antibodies (primarily IgG), purified and in human serum samples

Gold nanoholes

(~200 nm diameter and ~600 nm periodicity)

Recombinant antigen consisting of SARS-CoV-2 S protein or nucleocapsid (N) protein, both attached via noncovalent adsorption

-Antibody samples were incubated with antigen-functionalized surface for 1 h, followed by buffer rinsing and then endpoint imaging measurement based on transmitted light intensity at two fixed wavelengths.

-Developed intensity-based calibration curves for anti-S and anti-N antibodies in serum with detection range spanning 0.1 to 100 µg/mL.

-Demonstrated strong correlation between biosensing readouts and electrochemiluminescence (ECL) assay signal for analyzing clinical serum samples (convalescent, acute, control types).

-Detected higher anti-S antibody signals (88% sensitivity, 100% specificity) and anti-N antibody signals (95% sensitivity, 60% specificity) in convalescent/acute sera compared to control sera.

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SARS-CoV-2 neutralizing antibodies (primarily IgG) in human serum or plasma samples

Gold nanoholes

(~200 nm diameter and ~600 nm periodicity)

Recombinant antigens consisting of SARS-CoV-2 S protein, S protein RBD, membrane (M) protein epitopes, and N protein, all attached via noncovalent adsorption

-Antibody samples were incubated with antigen-functionalized surface for 30 min, followed by buffer rinsing and then endpoint imaging measurement based on transmitted light intensity at a fixed wavelength.

-Measured antigen-specific antibody measurements in 72 labeled clinical samples from individuals who were (1) naïve (no infection or vaccination), (2) vaccinated, (3) convalescent, or (4) convalescent and vaccinated.

-Convalescent and vaccinated group had strongest and broadest antibody responses while naïve group had lowest signals.

-Random forest-based machine learning model identified S and M antigens as most important predictors and was used to classify population-level COVID-19 infection rate (65% actual vs. 55% predicted) and vaccination rate (84% actual vs. 87% predicted) in 100 blind samples.

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SARS-CoV-2 neutralizing antibodies (primarily IgG), purified and in human serum samples

Gold nanoparticles (~35 or ~67 nm diameter) on a gold film

Recombinant antigens consisting of SARS-CoV-2 S1 protein, S protein RBD, and S protein-derived epitope peptide, all attached via noncovalent adsorption

-Antibody samples were incubated with antigen-functionalized gold film for 10–30 min followed by buffer washing and then gold nanoparticles were transferred on top prior to measurement.

-Validated sensing concept with 0-100 µg/mL antibody concentrations and significant difference in reflectance intensities for infected vs. healthy serum samples.

-Showed compatibility of sensing platform with portable, optical fiber reader (100% sensitivity, 99% specificity) and smartphone imaging (93% sensitivity, 97% specificity).

-Demonstrated ≥8-fold improved limit of detection for model antibody detection compared to immunofluorescence assay (IFA) formats.

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SARS-CoV-2 neutralizing antibodies (IgG and IgM), purified and in human serum samples (primarily IgG)

Gold nanoparticles (~35 nm diameter) on a gold film

Recombinant antigens consisting of SARS-CoV-2 S1 protein and S protein RBD from different variants, all attached via noncovalent adsorption

-Antibody samples were incubated with antigen-functionalized gold film for 10–30 min followed by buffer washing and then gold nanoparticles were transferred on top prior to measurement.

-Identified that S1 protein antigen and 5× diluted serum provided best detection performance, resulting in 100% sensitivity and 100% specificity.

-Determined that ~39% of blinded serum samples after COVID-19 pandemic had high neutralizing antibody levels whereas all prepandemic samples tested negative.

-Adapted measurement readout to high-throughput barcode format to enable variant-specific immunity profiling.

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SARS-CoV-2 neutralizing antibodies in human serum

Gold nanograins (~20-70 nm diameter) on gold film

Recombinant antigen consisting of SARS-CoV-2 S protein, attached via crosslinker

-Antibody samples were incubated with antigen-functionalized sensor chip for ~15 min followed by aqueous rinsing and then reflectance spectrum was measured.

-Total antibody levels in convalescent serum samples produced reflectance shifts in the 4–6 nm range and magnitude correlated with ELISA results.

-Total antibody levels in vaccinated serum samples generated 2–6 nm shifts and showed temporal fluctuations in line with vaccination schedule, which was supported by ELISA data.

-Developed sensor regeneration protocol based on pH-induced dissociation and facilitated consistent reflectance shifts in repeated fashion.

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SARS-CoV-2 neutralizing antibodies in human serum

Gold nanocups

(~220 nm diameter and ~440 nm periodicity)

Recombinant antigens consisting of SARS-CoV-2 S protein RBD from different variants, all attached via noncovalent adsorption

-Antibody samples were incubated with antigen-functionalized sensor chip for ~15 min and then ~5 min incubation with ACE2-functionalized Au@Pt nanoparticles, followed by reflectance spectrum measurement.

-50% inhibitory concentration (IC₅₀) values were calculated by determining how antibody dilution affects inhibition of surface-nanoparticle interaction (via RBD-ACE2 interaction).

-Measured IC₅₀ values of neutralizing antibodies to block ACE2 interaction with different RBD variants, including reduced efficacy against circulating mutants that agreed with plaque reduction neutralization test results.

-Assay had high sensitivity (94%) and specificity (100%) and did not demonstrate cross-reactivity with IgG antibodies against other respiratory viruses.

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ASFV antibodies (primarily p30-binding) in pig serum

Gold nanocups

(~200-300 nm diameter and ~400-500 nm periodicity)

Anti-pig IgG secondary antibody covalently attached to nanocup surface, recombinant p30 antigen attached to gold nanoparticles via noncovalent adsorption

-Antibody samples and p30 antigen-functionalized nanoparticles were mixed and added immediately to secondary antibody-functionalized sensor chip for 15-min incubation, followed by OD measurement at a fixed wavelength.

-Evaluation of 246 serum samples (ASFV-positive and negative) showed that the nanoplasmonic biosensing assay had around 96% sensitivity and 97% specificity, as validated by ELISA measurements.

-Dynamic range with quasi-linear response spanned 1:100 to 1:16000 serum dilutions and detection limit was comparable to that of indirect immunofluorescence.

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