Development of an immunosensor based on the exothermic reaction

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Development of an immunosensor based on the exothermic reaction between H2O and CaO using a common thermometer as readout Xiaoming Ma, Zhen Wang, Shan He, Chaoqun Chen, Fang Luo, Longhua Guo, Bin Qiu, Zhenyu Lin, Guonan Chen, and Guolin Hong ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00968 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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ACS Sensors

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Development of an immunosensor based on the exothermic

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reaction

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thermometer as readout

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Xiaoming Ma,a,b,c Zhen Wang,b Shan He,b Chaoqun Chen,c Fang Luo,c Longhua Guo,c

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Bin Qiu,c Zhenyu Linc*, Guonan Chenc, Guolin Honga*

between

H2O

and

CaO

using

a

common

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a. The department of laboratory medicine, the First Affiliated Hospital of Xiamen

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University, NO.55 Zhenhai Road, Siming District, Xiamen, Fujian 361005, China.

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b. School of Chemistry and Chemical Engineering, Key Laboratory of Organo-

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pharmaceutical Chemistry of Jiangxi Province, Gannan Normal University, Ganzhou,

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341000, P. R. China.

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c. MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian

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Provincial Key Laboratory of Analysis and Detection Technology for Food Safety,

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College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China.

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Corresponding author: Zhenyu Lin, Guolin Hong

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Email: [email protected], [email protected]

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Address: Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China.

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ACS Paragon Plus Environment

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Abstract

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Thermometer, one of the most commonly used instruments at home, is normally

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adapted to measure temperature directly with high accuracy, but rarely adopted to act

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as readout in the biosensors. It is necessary to find some way to establish a relationship

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between the concentration of the target and the temperature changing. In this study, a

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common thermometer was used as a readout to develop a convenient immunosensor.

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The designed immunosensor comprises of three components, including target

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recognition area, water flow system, and exothermic reaction bottle. The capture

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antibody for the target (carcinoembryonic antigen (CEA) was selected as a model target)

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was preloaded on the bottom of the recognition area. In the presence of CEA, a

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sandwich-type structure was formed between the capture antibody, CEA, and

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biotinylated detection antibody. Then the streptavidin functionalized platinum

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nanoparticles was labeled on the detection antibody due to biotin-avidin interaction.

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The captured platinum nanoparticles can effectively catalyze the decomposition of

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H2O2 into O2. The continuous production of gas resulted in pressure increment inside

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the reaction bottle and further pushed the water flow into the exothermic reaction bottle.

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Finally, the water reacted with calcium oxide to generate a large amount of heat in the

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exothermic reaction bottle. Thereby, the temperature inside the bottle was enhanced

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and recorded by a common thermometer easily. The temperature enhancement has a

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linear relationship with the CEA concentration in the range of 7.81 – 500 pg/mL with

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a detection limit of 0.6 pg/mL. Furthermore, by taking advantage of simplicity,

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compatibility, stability and high sensitivity, our temperature-based immunoassay has 2


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been applied to detect CEA in human serum samples with satisfactory results.

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KEYWORDS: Immunosensor, biosensor, cancer diagnosis, carcinoembryonic antigen,

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temperature, thermometer

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The thermometer is one of the most commonly used devices at home or in the

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laboratory. However, the applications of mostly commercialized thermometers are still

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limited in the temperature recording, and little attention had been paid on to act as a

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signal readout of biosensors. Herein, the key influencing factor is how to transfer the

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detection signal into temperature changing. To overcome these limitations, many

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research groups have made some useful attempt.1, 2 For examples, Gao’s group reported

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an exothermic chip for quantitative detection of limited heavy metal ions based on the

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analyte-hydrogel recognition, NaOH powders were served as an exothermic reagent

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and a forehead thermometer was used to measure the temperature variation.3 Fu’s group

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introduced a temperature-based measurement strategy based on the nanoparticle-

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mediated photothermal effect, where a near-infrared (NIR) laser was used to convert

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the assay signal into heat.4,

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green was hydrolyzed upon the addition of targets. The released dye converts excitation

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energy into heat under NIR-laser irradiation, thus allowing the construction of target-

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responsive thermometer.6 These results show that the temperature change in the

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detection system can be used as an important indicator for the biosensors construction.

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Owing to the antigen-antibody specific recognition system, immunoassay has

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good selectivity, and it can be applied to detect thousands of targets by simply changing

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the corresponding antigen-antibody pairs.7-9 To extend its application in the resource-

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limited area, the integration of many simple readout techniques (such as barometer,

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glucose meter, pH meter, naked eyes, and so forth ) with immunoassay have been

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developed for the determination of diverse targets.10-16 However, little attention has

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Alternatively, the liposome-encapsulated indocyanine

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been paid to couple with a typical thermometer as readout.

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In an early study, we developed a convenient and straightforward aptasensor based

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on converting molecular recognition into a weight variation with an electronic balance

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as signal readout.17 The amount of target enabled to be measured by weighing the

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discharged water. It is worth noting that self-heating food packaging, which is widely

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used in the food contents heating without external heat sources can generate a large

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amount of heat based on the exothermic reaction between calcium oxide (CaO) and

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water (H2O).18 Inspired by this principle, we considered integrating this commonly used

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exothermic reaction to construct temperature-based immunoassay. Theoretically, 10 L

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H2O reacts completely with CaO powder (2 g) can release 36.51 kJ energy under a

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closed adiabatic bottle (2.5 mL), which can lead to appropriate 12 °C increment of the

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air inside the bottle at room temperature. Therefore, a conventional thermometer with

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high accuracy can record the temperature changing of the system.

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Herein, we coupled this interesting self-heating reaction with an immunoassay to

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develop a simple but sensitive immunosorbent-based biosensor. A conventional

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thermometer was used as the signal readout for the sensitive detection of

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carcinoembryonic antigen (CEA) (chosen as the model target). The temperature

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increment was found to be linearly proportional to the concentration of CEA,

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establishing the quantitative determination of CEA in the human serum samples with

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satisfactory results.

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EXPERIMENTAL SECTION

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Preparation of the capture antibody immobilized glass vial and streptavidin 5


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functionalized platinum nanoparticles.

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The capture antibody immobilization on the glass vial was performed according to the

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previous report.19 Besides, the streptavidin functionalized platinum nanoparticles was

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synthesized based on our previous study.17 The detail procedures are presented in

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supporting information.

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Design and fabrication of the immunosensor using thermometer as readout.

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The proposed biosensor comprises a target recognition area, water flow system,

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and exothermic reaction bottle, which separately manufactured and assembled once

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used (Figure S1). The conventional immunoassay procedure was performed in the

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target recognition area. After the enzyme catalysis decomposition of H2O2 into H2O and

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O2, the pressure inside the bottle (10 mL) dramatically increased. A stop-cocks was

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fabricated to control the water flow from the reservoir into the exothermic reaction

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bottle. Meanwhile, the CaO powder which preloaded inside the exothermic reaction

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bottle (2.5 mL) assembled with a common thermometer reacted with the H2O, and the

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temperature increment can be recorded by a common thermometer. For the sensitivity

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determination of CEA, the stop-cocks was opened after H2O2 decomposition reaction

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of 15 min. Thus the suddenly increased pressure pushes the water flows from the target

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recognition bottle into the exothermic reaction bottle to generated temperature

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increment.

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Analytical procedure of the detection.

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Firstly, 200 µL aliquots of CEA samples in sample dilution buffer (50 mM Tris,

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0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) were added to the capture antibody 6


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immobilized glass vial and incubated for 2 h at room temperature. Each glass vial was

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washed three times with 500 µL wash buffer (50 mM Tris, 0.14 M NaCl, 0.05% Tween

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20, pH 8.0), then removed any remained wash buffer via inverting the glass vial and

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blotting it against clean paper towels after the last wash. The biotinylated detection

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antibody (100 µL, 1.0 µg/mL) in dilution buffer was added and incubated for 1 h at

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37 °C. The washing step was repeated, and then PtNPs-streptavidin conjugation (100

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µL, 0.5 nM) was added with incubation for 30 min at room temperature. After repetition

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of washing steps, H2O2 (1 mL, 30 wt%) was added to the vial and sealed the twist cap

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immediately, while the stop-cocks was closed at this moment. The bound PtNPs would

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catalyze the dissociation of H2O2 to H2O and O2, leading to the increment of pressure

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inside the bottle. After the catalysis reaction for 15 min, the stop-cocks was opened.

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The generated pressure difference would finally push the water flow into the

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exothermic reaction bottle. The resulting exothermic reaction of CaO dissolve in the

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water was monitored by a common thermometer after 5 min of reaction time, and the

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temperature increment was readout by the naked eye. The sensitivity of this

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temperature-based sensing platform can be regulated by adjusting the enzymatic

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reaction time of PtNPs. To meet the requirement of actual sample determination, we

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control the catalysis reaction time at 5 min.

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RESULTS AND DISCUSSION

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Principle of immunosensor using a thermometer as readout.

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The principle of the proposed immunosensor using a common thermometer as

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readout is shown in Scheme 1. The mouse anti-CEA monoclonal antibody (act as the 7


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capture antibody) had been preloaded on the bottom of a glass vial firstly. The

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biotinylated rabbit anti-CEA polyclonal antibody was used as the detection antibody.

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Upon the addition of target (CEA in this study), a sandwich-type structure will form

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between capture antibody-CEA-detection antibody. Then the PtNPs-SA was modified

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on the sandwich structure through biotin-streptavidin interaction. After repeating

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washing steps to eliminate the excess unbounded PtNPs-SA, the glass vial was sealed

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immediately after the addition of H2O2. The pressure inside the vial increased gradually

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as the decomposition of H2O2 by PtNPs-SA when the stop-cocks was closed. Once the

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stop-cocks was turned on, a certain amount of water would flow into the exothermic

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reaction bottle immediately due to the pressure difference. The reaction between water

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(H2O) and the calcium oxide (CaO, which was preloaded in the exothermic reaction

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bottle) produces a large amount of heat through the following reaction: CaO (s) + H2O

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(l)  Ca(OH)2; H 65.2 kJ·mol-1. This enormous heat will induce the temperature

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enhancement of the system and which can be detected by a common thermometer easily.

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Thus, the relationship between temperature enhancement and the target concentration

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can be established successfully. Based on this principle, a simple immunosensor was

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designed using a simple thermometer as readout.

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Scheme 1. Principle of the temperature-based immunosensor using a common

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thermometer as readout.

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Characterization of the PtNPs-streptavidin conjugates.

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Compared with catalase enzyme, PtNPs can effectively catalyze the breakdown of

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H2O2 to generate abundant O2 without the apparent deactivation, inducing a significant

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increase in pressure of the sealed container.20 To improve the versatility of PtNPs in the

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application of immunoassay, PtNPs had been conjugated with streptavidin, and the

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synthetic process was characterized by UV-vis absorption assay. As shown in Figure

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S2(A), the absorbance of H2PtCl6 shifts from 259 to 300 nm after the incubation with

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ascorbic acid, and the color of the solution change from light yellow to brown,

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suggesting the successful synthesis of PtNPs. Then the prepared PtNPs had been

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conjugated with streptavidin (SA), the UV-vis absorbance spectra show no significant

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change. TEM and DLS assays are performed to confirm whether the SA had been

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successfully modified on the PtNPs. The hydrodynamic diameter increase from 72 to

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88 nm, and the zeta potential increase from 40.6 to 34.9 mV after the conjugation

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(Figure S3). These results indicate that the PtNPs-SA conjugates had been well-

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synthesized. The TEM image of the dendritic nanoparticles (Figure S2B) indicates that 9


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the nanoparticles have good dispersion and uniform size with an average diameter 25

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nm, thus allowing its further modification on the biotin-conjugated antibody.

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To study the suitability of PtNPs-SA in the catalytic decomposition of H2O2, we

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compared its catalytic performance with catalase enzyme through the drainage

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experiment. A certain amount of PtNPs-SA or catalase was added into the 30% H2O2

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solution to initiate the gas-generation reaction. A large amount of oxygen produced

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base on the catalysis reaction result in a significant pressure increase, thereby a certain

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amount of water flowed to exothermic bottle as a result. The amount of discharged

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water can be recorded by an electronic balance. As shown in Figure 1(A) and (B), both

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PtNPs-SA and catalase can push the movement of water flow as a function of time, this

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phenomenon is induced solely by the O2 generated from the decomposition of H2O2.

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However, catalase enzyme fails to catalyze the O2 generation after 5 min. Moreover,

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the catalysis efficiency of catalase would dramatically decrease under high H2O2

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concentration (data not shown), which can be attributed to the destructive effect of

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catalase under high concentration of H2O2.21 In comparison to nature catalase, the

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PtNPs-SA shows increased catalytic ability with increasing H2O2 concentration and

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remains steadily catalytic activity over a long period of time. The catalysis process of

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PtNPs-SA toward H2O2 is a zero-order reaction, so the amount of the discharged water

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increased almost linearly with the extension of the reaction time.19 Furthermore, the

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amount of water has a direct linear relationship with the concentration of PtNPs-SA

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(Figure S4). Therefore, we can meet the specific requirement of sensitivity by adjusting

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the reaction time and PtNPs-SA amount. 10


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Figure 1. (A) Catalytic decomposition of H2O2 with different concentrations of PtNPs-

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SA in 30 wt% H2O2 at different times, the concentration of PtNPs-SA from a to f were

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10, 25, 50, 100, 150, 200 pM, respectively; (B) Catalytic decomposition of H2O2 with

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different concentrations of catalase in 30 wt% H2O2, the concentration of catalase from

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a to e were 2.5, 5, 10, 15, 20 nM, respectively.

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Measurement of an exothermic reaction.

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A small amount of water reacts completely with CaO powder (2.0 g) can lead to a

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huge temperature increment in a closed adiabatic bottle (2.5 mL) in theoretically.

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However, the actual temperature increment measured by our method is much lower

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than that of the theoretical calculation (shown in Figure 2A). The main reason lies in

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that the external thermal absorption from the reaction bottle and reagents (such as CaO,

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Ca(OH)2 and so forth). As shown in Figure 2(B), the temperature increases gradually

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as a function of reaction time after mixing the CaO with H2O, and finally reach the

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maximum value at 300 s (5 min). Moreover, the temperature increment enhances with

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the increasing amount of water added. Therefore, it is possible to utilize these

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phenomena for further study.

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Figure 2. (A) Comparison of the theoretical and actual temperature changing values

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after the reaction of different amounts of water with the CaO powder (2 g); (B)

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Temperature increment with different amounts of water (50 L, 150 L, 250 L)

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reacted with the CaO powder (2 g) at different times.

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Performance of the temperature-based immunosensor.

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The variation of temperature responses recorded by a common thermometer with

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different concentrations of CEA (the stop cock requires opened after 15 min of H2O2

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decomposition reaction) is shown in Figure 3(A). The degree of temperature increment

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has a linear relationship with CEA concentration in the range from 7.81 pg/mL to 500

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pg/mL (Figure 3 B) with a correlation coefficient of 0.994. The regression can be

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expressed as follows:

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T = 0.1464 C(CEA) + 0.4828

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where the temperature increase (T) defines as the temperature change of the bottle

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before and after the exothermic reaction, C(CEA) is the concentration of CEA. The

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limit of detection is 0.6 pg/mL (defined as three standard deviations above the mean

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background signal), which is more sensitive compared to that of some previous reports

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(Table S1)22-28. Thus, the proposed sensing platform can sufficiently satisfy the need 12


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for practical applications in the areas of daily life.

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Figure 3. (A) Optical images of temperature increment recorded by the thermometer.

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The concentrations of CEA from left to right were 7.81, 15.6, 31.2, 62.5, 125, 250, 500

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pg/mL, respectively; (B) A plot of the temperature increase (T) versus the

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concentration of CEA in the sample.

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To evaluate the specificity of the proposed biosensor for CEA detection, some

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common proteins present in the serum was selected as negative controls, including

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human serum albumin (HSA), immunoglobulin G (IgG), prostate-specific antigen

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(PSA), and hepatitis B surface antigen (HBsAg). As shown in Figure 4, a significant

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temperature increment is recorded in the presence of CEA (150 pg/mL), but nearly no

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temperature changing is observed even the concentration of interferents (1500 pg/mL)

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was ten times than that of CEA. Therefore, these outcomes indicate that our proposed

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strategy has high selectivity in the detection of CEA, which lies in the inherent

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specificity between antigen and antibody. Furthermore, the relative standard deviation

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(RSD) was determined to be 4.6% for three measurements (the concentration of CEA

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was 500 pg/mL), which confirms that the as-proposed biosensor is reproducible for 13


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CEA determination.

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Figure 4. The selectivity of the proposed biosensor for CEA detection. The

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concentrations of HSA, IgG, PSA, HBsAg, CEA were 1500 pg/mL, 1500 pg/mL, 1500

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pg/mL, 1500 pg/mL and 150 pg/mL, respectively.

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The long-term stability and the inference of ambient temperature variation were

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tested to evaluate the stability of the temperature-based immunoassay for CEA

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detection. As shown in Figure S5, the temperature signal has a negligible change within

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20 days, which can attribute to the good airtightness of our designed device. However,

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temperature variation response was change after 20 days, it mainly due to the inference

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of CO2 and humidity variation. Therefore, to improve the long-term stability, a suitable

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airtightness device and dry storage environment are desired. It can be found that this

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assay is relatively stable at different ambient temperatures (Figure S6). However, the

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signal response was increased when the ambient temperature higher than 35 °C. This

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phenomenon mostly due to the self-decomposition of H2O2 and the increased catalysis

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ability of PtNPs under a high-temperature environment.

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Real sample analysis. 14


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To investigate the accuracy of the proposed immunosensor for CEA determination

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in the practical application, we tested 15 serum samples (donated by the volunteers)

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obtained from the local hospital (Fuzhou, China). The serum samples with unknown

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CEA concentrations were monitored by the temperature-based testing and compared

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these results with that obtained from the hospital using chemiluminescence

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microparticle immunoassay (CMIA), a current “gold standard” strategy of practical

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detection of CEA in hospitals. As shown in Figure 5(A), results of both methods are

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strongly correlated, showing a linear correlation of y = 1.021x – 1.582 (where y denotes

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the detection value by CMIA method, x denotes the detection value by the proposed

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method) with a correlation coefficient of 0.99. This result reveals that a good

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consistency is observed between the two methods. In addition, the Bland-Altman plots

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were constructed to evaluate the agreement between the proposed temperature-based

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sensing platform and conventional CMIA assay. As shown in Figure 5B, the proposed

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sensing platform with temperature readout shows a bias offset of –0.5 μg/mL at the

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limit of agreement (bias  1.96 SD) from –18.8 to 17.9 μg/mL for CEA detection

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compared to the CMIA method. Based on these results, the accuracy of this

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temperature-based method is nearly the same as that of the conventional CMIA method,

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indicating the possibility of potential application in the clinical diagnosis.

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Figure 5. (A) Correlation of the proposed method results with the standard clinical

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results for CEA detection in 15 serum samples; (B) Bland-Altman analysis for the

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difference between the proposed method with CMIA for CEA detection in 15 clinical

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samples.

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CONCLUSION

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In summary, a temperature-based immunosensor using an ordinary thermometer

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as readout had been established in this study. The designed biosensor successfully

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transduces the antibody-antigen recognition event into the temperature change with a

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common thermometer as signal readout. By taking advantages of high catalysis

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efficiency of nanozyme and self-heating reaction between CaO and H2O, the

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quantitative results of CEA can be obtained directly without the assistant of any

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expensive instruments or power sources other than a simple thermometer. With the

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merits of high sensitivity, equipment-free, and visual quantitative readout, the as-

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proposed strategy lay the foundation of thermo-based biosensors applications for

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diverse targets in the resource-limited area. Besides, materials with higher thermal

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transformation efficiency or naked-eye visualization thermochromism property can 16


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select as a candidate for further temperature-based biosensor design.

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ACKNOWLEDGMENT

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This work was financially supported NSFC (21804022, 21575025, and 2155027),

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the Program for Changjiang Scholars and Innovative Research Team in University (No.

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IRT15R11) and the cooperative project of production and study in University of Fujian

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Province (2018Y4007), the Sciences Foundation of Fujian Province (2018J01685,

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2018J01682), STS Key Project of Fujian Province (2017T3007), and the Science and

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Technology Project of the Education Department of Jiangxi Province of China (No.

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GJJ170846).

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ASSOCIATED CONTENT

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Supporting Information (SI) is available free of charge via the Internet at

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http://pubs.acs.org. (Materials, apparatus, experimental details, additional images and

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tables)

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AUTHOR INFORMATION

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Corresponding Authors

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*E-mail: [email protected] (G.H.)

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*E-mail: [email protected] (Z.L.)

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ORCID

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Xiaoming Ma: 0000-0001-6875-8289

313

Fang Luo: 0000-0001-7495-450X

314

Longhua Guo: 0000-0003-0706-0973

315

Zhenyu Lin: 0000-0001-7890-6812 17


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Notes

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The authors declare no competing financial interest.

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Development of an immunosensor based on the exothermic reaction

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