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Cite This: Anal. Chem. 2019, 91, 8274−8279
Stable and Photothermally Efficient Antibody-Covered Cu3(PO4)2@Polydopamine Nanocomposites for Sensitive and CostEffective Immunoassays Xiaofeng Tan, Xiaoying Wang,* Lianhua Zhang, Luyao Liu, Gengxiu Zheng, He Li,* and Feimeng Zhou*
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Institute of Surface Analysis and Chemical Biology, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China S Supporting Information *
ABSTRACT: Polydopamine (PDA)-coated or encapsulating Cu3(PO4)2 (Cu3(PO4)2@PDA) nanosheets were synthesized, allowing the C-reaction protein (CRP) antibody to be attached electrostatically for immunosensing of CRP with simple photothermal detection. The antibody-covered Cu3(PO4)2@PDA nanosheets replace the antibody-conjugated enzyme in the enzymelinked immunosorbant assays. Owing to the high surface area of the 2-D-structured Cu3(PO4)2@PDA nanosheets and the coabsorption of light in the near-IR spectrum by Cu3(PO4)2 and PDA, a small amount of Cu3(PO4)2@PDA confined in the wells of a titer plate generates an easily detectable temperature change after irradiation at 808 nm. The temperature changes, measured by an inexpensive pen-type thermometer, increased linearly with the analyte concentration from 0.42 to 16 pM. We found that the linear relationship can be fitted by the isotherm derived from responses collected from heterogeneous sensors covered with different ligand or antibody densities. The low detection limit (0.11 pM) is largely due to the attachment of a great number of antibodies onto the flat nanosheets. The antibody-covered Cu3(PO4)2@PDA nanosheets are stable and can be used under conditions that are generally unfavorable to enzymatic activities. The excellent agreement between our results and immunoturbidimetric assays of CRP in serum samples from patients and healthy donors demonstrates its utility for disease diagnosis in clinical settings. This cost-effective, biocompatible, and convenient photothermal immunosensor affords a range of possibilities for detecting diverse protein biomarkers.
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terials.16,17 Under illumination, a miniscule amount of photothermal nanomaterials confined in or released into a small cavity generates a sizable change in the solution temperature, which can be detected by a simple and inexpensive device such as a miniature thermometer. Photothermal compounds or nanomaterials can effectively transfer energy from the incident light (typically a laser beam) into heat, enabling photothermal therapy18,19 and many other applications.20,21 A photothermal effect-based immunoassay was first demonstrated by Li and co-workers through the use of the antibody-coated Fe3O4 nanoparticles.22 The photothermal effect can be further enhanced via dissolution and subsequent conversion of the Fe3O4 nanoparticles to Prussian blue nanoparticles. In a more recent report, a near IR-laser was used to generate solution temperature changes by irradiating indocyanine green released from the interior of liposomes during liposome hydrolysis by target-responsive DNAphospholipase conjugates.23 Thus far, theoretical models or
nzyme-linked immunosorbent assay (ELISA) has been widely recognized as a gold standard for clinical assays of protein biomarkers.1−4 However, conjugation of enzymes to antibodies not only complicates the reagent preparation and increases the assay cost but also leads to uncertainties stemming from the decrease or complete loss of enzymatic activities.5 To address potential enzyme degradation and retain enzymatic activities in storage and during the assay, endeavors have been embarked on to bind robust enzymes onto secondary antibodies via efficient cross-linking methods.4,6,7 Meanwhile, reporter molecules or nanomaterials capable of amplifying detection signals have been synthesized to replace enzymes used in conventional ELISA.8 The use of such “nanozymes”9−11 is particularly appealing because of their low cost, high stability, and long durability. Another attractive feature of nanozymes is that the immunoassay does not require any modification of the ELISA equipment (e.g., titer plate readers) and procedures.12,13 These nanomaterial-based “nanozymes” allow measurements be performed under experimental conditions (e.g., elevated temperatures and nonphysiological pH values) that are harsher than those employed for ELISA or immunoturbidimetric assays.8,14,15 A type of nanoconjugate that has recently attracted attention is photothermal nanoma© 2019 American Chemical Society
Received: February 21, 2019 Accepted: June 10, 2019 Published: June 10, 2019 8274
DOI: 10.1021/acs.analchem.9b00968 Anal. Chem. 2019, 91, 8274−8279
Article
Analytical Chemistry
ing to the literature procedure33 with some modifications. Briefly, 500 mg of CuSO4·5H2O was dissolved in 25 mL of glycol and mixed with 20 mL of water solution containing 132 mg of diammonium hydrogen phosphate to produce a clear aquamarine solution. The mixture was transferred into a hydrothermal reactor, which was then sealed and maintained at 120 °C for 4 h. After the hydrothermal synthesis, a blue solid was obtained when the solution was cooled to room temperature. The Cu3(PO4)2 nanosheets collected after centrifugation (8000 rpm for 10 min) were washed with deionized water and alcohol and dried in vacuo for 10 h. With vigorous stirring, dopamine hydrochloride (10 mg) was added into 5 mL of Tris-HCl buffer (pH 8.5) containing dispersed Cu3(PO4)2 nanosheets (1 mg/mL), and the stirring continued for 2 h. The Cu3(PO4)2@PDA nanosheets were obtained after the solution color turned from pale yellow into black. The Cu3(PO4)2@PDA nanosheets were collected upon centrifugation (8000 rpm) for 10 min and washed with deionized water three times. To cover the Cu3(PO4)2@PDA nanosheets with Ab2, redispersion of the Cu3(PO4)2@PDA (5 mg) nanosheets in 5 mL of PBS buffer (pH 7.4) was followed by addition of 1 mL of Ab2, anti-CRP (1 μg/mL). The solution was then incubated for 6 h under agitation. After centrifugation and washing with PBS buffer, the Cu3(PO4)2@PDA−Ab2 solution was stored at 4 °C. Photothermal Immunoassay. The anti-CRP-Ab1 antibody (1 μg/mL, 200 μL) was added into the wells of a 96 titer plate and incubated at 4 °C for 10 h. Nonspecific binding sites were blocked with BSA (w/v = 1:100, 200 μL, 1 h). Aliquots (200 μL) of different CRP solutions were added into the wells and allowed to stand for 1 h. Finally, 200 μL of Cu3(PO4)2@ PDA−Ab2 (0.5 mg/mL) was added into the wells, and the titer plate was shaken vigorously at room temperature for 30 min. Between steps, the wells were washed with PBS buffer three times. Near IR irradiation was implemented using a continuous-wave diode laser (MW-GX-808/1−5000 mW) centered at 808 ± 5 nm with a 1 W output power (Changchun Laser Optoelectronics Technology Co., Changchun, China). Under irradiation at 0.71 W cm−2 for 10 min, the light-to-heat capacity values of Cu3(PO4)2/PDA and Cu3(PO4)2 (500 μL) in 1.5 mL centrifuge tubes were measured using an inexpensive (
