Improving the sensitivity of immunoassays by reducing non-specific binding of poly(acrylic acid) coated upconverting nanoparticles by adding free poly(acrylic acid)

Abstract

Upconverting nanoparticles (UCNPs) are attractive reporters in immunoassays because of their outstanding detectability. However, non-specific binding of antibody-UCNP conjugates on protein coated solid support results in background, which limits the immunoassay sensitivity. Thus, the full potential of UCNPs as reporters cannot be fully exploited. The authors report here a method to improve the sensitivity of UCNP-based immunoassays by reducing the non-specific binding of antibody-UNCP conjugates on the protein coated solid support. In the assays studied here, poly(acrylic acid) (PAA) coated NaYF:Yb,Er type UCNPs were conjugated to two different antibodies against cardiac troponin I (cTnI) and thyroid stimulating hormone (TSH). The two-step heterogeneous sandwich immunoassays were performed in microtitration wells, and the green luminescence of antibody-UCNP conjugates was measured at 540 nm upon 980 nm excitation. Non-specific binding of antibody-UCNP conjugates was reduced by mixing free PAA with PAA coated UCNPs before adding the UCNPs to the wells. The free PAA in the buffer reduced the background in both cTnI and TSH immunoassays (compared to the control assay without free PAA). The limits of detection decreased from 2.1 ng·L to 0.48 ng·L in case of cTnI and from 0.070 mIU·L to 0.020 mIU·L in case of TSH if PAA is added to the buffer. Presumably, the effect of free PAA is due to blocking of the surface areas where PAA coated UCNP would bind proteins non-specifically. The method introduced here is likely to be applicable to other kinds of PAA-coated nanoparticles, and similar approaches conceivably work also with other nanoparticle coatings. Graphical abstract The presence of free poly(acrylic acid) (PAA) in a buffer solution prevents aggregation and non-specific protein binding of PAA-coated upconverting nanoparticles (UCNPs) in heterogeneous sandwich immunoassays. The decrease in non-specific binding enables distinctly more sensitive assays to be performed.

Effect of Particle Size and Surface Chemistry of Photon‐Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

“…The conjugation of mAb 625cc and mAb 560cc was adapted from a previously published protocol [28] and all steps were performed at RT. A dispersion of 250 μL of PAA-coated UCNPs (2 mg) in 20 × 10 −3 m aqueous MES buffer (pH 6.1) was activated using 20 × 10 −3 m EDC and 30 × 10 −3 m sulfo-NHS for 45 min under shaking.…”

“…In another study, we applied PAA-coated UCNPs to the detection of cTnI in the analog mode, which resulted in an LOD of 0.48 pg mL −1 . [28,29] Here, we employ both surface modification strategies for UCNPs and critically assess the effect of the UCNP label size on the performance of the analog and digital immunoassay for cTnI. The schemes of the sandwich ULISAs using either type of label are shown in Figure 1.…”

Polymeric nanoparticles in the diagnosis and treatment of myocardial infarction: Challenges and future prospects

“… Diagnostic tool Type of nanosystem Entrapped cargo Surface modification Diagnostic effect Ref. CK-MB immunochromatographic assay Polystyrene microparticles Europium (III) chelate Carboxylate Rapid simple quantitative detection of CK-MB levels [ 24 ] No requirement for large volumes of serum Range of detection: 0.85–100.29 ​ng/mL Limit of detection: 0.029 ​ng/mL cTnI immunoassay Polystyrene/Poly (acrylic acid) core-shell NPs Red fluorescent dye “Nile Red” Anti-cTnI antibodies – Effective for early screening and diagnosis of acute MI [ 29 ] Inexpensive, accurate rapid detection of cTnI levels Range of detection: 0–40 ​ng/mL Limit of detection: 0.016 ​ng/mL Upconverting NPs Lanthanide ions Anti-cTnI antibodies Poly (acrylic acid) Effective for diagnosis of MI [ 134 ] Sensitive detection of cTnI levels Range of detection: 0–100 ​ng/mL Limit of detection: 0.00048 ​ng/mL Cardiac magnetic resonance Human serum albumin NPs Gadolinium-diethylenetriamine-pentaacetic acid Transferrin Allowed loading of lower doses of Gadolinium [ 135 ] Improved Gadolinium enhancement and circulation time Enhanced imaging of cardiac muscle via transferrin-mediated targeting Bovine serum albumin NPs MnO 2 motifs Low pH-sensitive motifs Enhanced T1 relaxivity [ 136 ] Induced rapid NPs accumulation, increased contrast enhancement in infarcted myocardium of MI rabbits Improved diagnostic accuracy compared to Gd-DTPA Polymeric nanoreactor Acid phosphatase Glucose-1-phosphate Gadolinium chloride Polyguanylic acid …”

Point-of-Care Compatibility of Ultra-Sensitive Detection Techniques for the Cardiac Biomarker Troponin I—Challenges and Potential Value

“…Over a quarter of the papers reviewed employed a luminescence detection method, the vast majority of which opted for fluorescence or ECL detection. However, there are exceptions, most notably the development of an immunoassay which relies on the emission of visible light from upconversion luminescence (UCL) to detect cTnI [117]. UCL has the distinct advantage of a reduced auto-fluorescence background signal in comparison to some other luminescence methods, in addition to utilising nanoparticles that absorb near-infrared light and emit visible or UV light [118].…”

“…Figure 3 and Figure 4: [37,38,48,49,50,57,58,61,65,66,68,69,70,71,74,84,85,86,87,88,89,93,94,95,96,97,104,105,111,112,115,116,117,120,121,122,123,125,126,127,128,130,132,133,134,136,138,144,145,148,150,151,159,161,174,175,176,184,185,186,187,188,189,190,…”