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Stable and photothermally efficient antibody-covered Cu(PO)@polydopamine nanocomposites for sensitive and cost-effective immunoassays Xiaofeng Tan, Xiaoying Wang, Lianhua Zhang, Luyao Liu, Gengxiu Zheng, He Li, and Feimeng Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00968 • Publication Date (Web): 10 Jun 2019 Downloaded from http://pubs.acs.org on June 11, 2019

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Analytical Chemistry

Stable and photothermally efficient antibody-covered Cu3(PO4)2@polydopamine nanocomposites for sensitive and cost-effective immunoassays Xiaofeng Tan, Xiaoying Wang*, Lianhua Zhang, Luyao Liu, Gengxiu Zheng, He Li*, Feimeng Zhou*

Institute of Surface Analysis and Chemical Biology, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT:

Polydopamine

(PDA)-coated

or

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encapsulating

Cu3(PO4)2

(Cu3(PO4)2@PDA) nanosheets were synthesized, allowing the C-reaction protein (CRP) antibody 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 enzyme-linked immunosorbant assays. Owing to the high surface area of the 2-D-structured Cu3(PO4)2@PDA nanosheets and the co-absorption 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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INTRODUCTIONS Enzyme-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 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 nanomaterials-based ‘nanozymes’ allow measurements be performed under experimental conditions (e.g., elevated temperatures and non-physiological pH values) that are harsher than those employed for ELISA or immunoturbidimetric assays.8,14,15 A type of nanoconjugates that have recently attracted attentions is photothermal nanomaterials.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


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liposome hydrolysis by target-responsive DNA-phospholipase conjugates.23 Thus far, theoretical models or adsorption isotherms for target attachment onto photothermal nanomaterials or constructs have not been formulated to shed light on the response-concentration relationship. Furthermore, we envision that, in designing nanozymes based on the photothermal effect, choosing materials whose inherent photothermal conversion efficiencies (PCEs) are high should be paired with tailoring the nanomaterials into a shape that absorbs light most efficiently. In the latter aspect, 2-D nanomaterials (i.e. nanosheets or nanoflakes) should be more advantageous than their 3-D counterparts (i.e. nanoparticles or nanotubes) as they render a much larger light-absorbing surface. In this work, we coated photothermally efficient Cu3(PO4)2 nanosheets with a thin layer of polydopamine (PDA) and used the resultant Cu3(PO4)2@PDA nanosheets for highly sensitive photothermal immunoassay of C-reaction protein (CRP). We selected Cu3(PO4)2 nanosheets based on the following considerations: (1) the aforementioned favorable light-absorbing property of 2-D structures;24 (2) biocompatibility and biodegradability (e.g. phosphate compounds are used for hard tissue repairs);25,26 and (3) versatility for preparing a wide range of nanomaterials such as organic-inorganic nanoflowers for subsequent coating with other materials.27-29 We should note that Cu3(PO4)2 is also capable of enhancing the activity and stability of antibodies,30 an added advantage as far as conjugation of a secondary antibody for ELISA is concerned. Polydopamine (PDA), capable of absorbing strongly in near IR, is also biocompatible, owing to its inertness in neuromelanin, the insoluble deposit in the substantia nigra of the brain.31 Coating the Cu3(PO4)2 nanosheets with the positively charged PDA film offers a convenient way to electrostatically attach the secondary antibody of CRP, the model analyte used in our proof-of-concept assay. Compared to other existing methods used in clinical assays of CRP (e.g. immunoturbidity and nephelometry15,32), our method drastically cuts down the assay cost without compromising the high sensitivity inherent in the use of an enzyme-antibody conjugate.


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MATERIALS AND METHODS Reagents. Copper sulfate pentahydrate (CuSO4·5H2O) and diammonium hydrogen phosphate were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). C-reaction protein (CRP) and its primary (Ab1) and secondary (Ab2) antibodies and bovine serum albumin were obtained from Shanghai Linc-bis Science Co., Ltd. (Shanghai, China). Dopamine hydrochloride was acquired from Aladdin Industrial Corp. (Shanghai, China). Glycol was obtained from Tianjin Damao Chemical Reagent Co., Ltd. (Tianjin, China). Other chemicals were of analytical grade and used as received. Material Characterization. Morphological characterizations were performed on a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) and an Asylum MFP-3D atomic force microscope (Santa Barbara, CA, USA). X-ray diffraction (XRD) analyses were performed on a Rigaku D/MAX 2200 X-ray diffractometer (Tokyo, Japan) with 2 scanned between 10° and 80°. FT-IR spectra were collected in the range of 400 4000 cm

1

on a JASCO FT-IR-410 spectrometer (Tokyo, Japan).

UV-vis measurements were conducted on an Agilent Cary 500 UV-Vis-NIR spectrometer (Santa Clara, CA, USA). Synthesis of Cu3(PO4)2 and Cu3(PO4)2! " # $2 Nanosheets. Cu3(PO4)2 nanosheets were synthesized according to literature procedure33 with some modifications. Briefly, 500 mg CuSO4·5H2O was dissolved in 25 mL glycol and mixed with 20 mL water solution containing 132 mg 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 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 ACS Paragon Plus Environment

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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 PBS buffer (pH 7.4) was followed by addition of 1 mL Ab2, anti-CRP (1 M J

/9 The solution was then

incubated for 6 h under agitation. After centrifugation and washing with PBS buffer, the Cu3(PO4)24+. N 82 solution was stored at 4 °C. Photothermal Immunoassay. The anti-CRP-Ab1 antibody (1 M J

200 M / 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 ML, 1 h). Aliquots (200 M / of different CRP solutions were added into the wells and allowed to stand for 1 h. Finally, 200 M Cu3(PO4)24+. N 82 (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 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 M / in 1.5-mL centrifuge tubes were measured using an inexpensive (< $4) EW 300 pen-type digital thermometer (Zhengzhou Boyang Instrument Co., Zhengzhou, China). Immunoassays were performed in 96 titer plates under the same irradiation power for 1 min, at ambient temperature (between 25 °C and 26 °C). Serum Samples Assay. Serum samples (obtained from Urology Department, Renji Hospital of Shanghai Jiao Tong University School of Medicine) were diluted 1000 times with PBS (pH 7.4) before use. The procedure for serum samples assay is the same as that used to obtain the calibration curve. Five samples (two from patients and three from healthy donors) were tested.

Immunoturbidimetric assays were

performed at Renji Hospital. A student’s t test was used to compare the differences of between these two methods.

RESULTS AND DISCUSSION ACS Paragon Plus Environment

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nanosheet and UV-vis spectra of the Cu3(PO4)2 (black curve) and Cu3(PO4)2@PDA (red) nanosheets are presented in panels (E) and (F), respectively. Inset of panel F shows the colors of the Cu3(PO4)2 (left vial) and Cu3(PO4)2@PDA nanosheets (right vial).

Figure 2A and 2B are representative TEM images of the Cu3(PO4)2 and Cu3(PO4)2@PDA nanosheets, respectively. Most nanosheets are disk in shape, with diameters in the range of 67.3 ± 12.3 nm. Such a dimension is consistent with those reported in the literature.24 Comparison of the two TEM images reveals that the PDA coating did not change the apparent morphology and dimension of the Cu3(PO4)2 nanosheets, suggesting that the PDA coating is extremely thin. Figure 2C and 2D are AFM images of the Cu3(PO4)2 and Cu3(PO4)2@PDA nanosheets showing detailed morphologies and thicknesses. The Cu3(PO4)2@PDA nanosheets (2.9 ± 0.2 nm) are significantly thicker than the Cu3(PO4)2 counterparts (1.5 ± 0.3 nm) and are more dispersible, due to the coating of the positively charged PDA films.34 The X-ray diffraction (XRD) pattern depicted in Figure 2E has the characteristic peaks that can be well-indexed to Cu3(PO4)2·3H2O (JCPDS card no 22-0548). The XRD pattern of the Cu3(PO4)2@PDA nanosheets are essentially the same (data not shown). The UV-vis spectrum of Cu3(PO4)2 (black curve in panel F) does not exhibit any absorption peaks before 400 cmN1 and the solution color (left panel in the inset) is pale blue. The Cu3(PO4)2 @PDA (red curve) solution is black (right panel of the inset) and there is a small and broad peak between 350 and 650 nm in the UV-vis spectrum (black curve). It is also obvious that the absorbance of the Cu3(PO4)2@PDA nanosheets at 808 nm is considerably greater than that of the Cu3(PO4)2 counterpart in Figure 2F, a result of additional absorption by the PDA thin film.35,36


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Figure 3. FT-IR spectra of Cu3(PO4)2 (black) and Cu3(PO4)2@PDA (red) nanosheets.

We carried out FT-IR analyses to further confirm the successful functionalization of Cu3(PO4)2 with PDA. In Figure 3, the characteristic stretching and bending peaks of the phosphate P O bonds appeared in the ranges of 940 1100 cm cm

1

1

and 560 627

(black curve). The peak at around 3400 cmN can be attributed to the N–H

vibration, and that at 1510 cm

1

can be ascribed to the stretching vibration of indole.

These peaks are indicative of the presence of the PDA coating.37-39

Figure 4. (A) Temperature-irradiation time curves recorded from solutions comprising 0, 30, 60, 100, and 500 M J

Cu3(PO4)2@PDA nanosheets. (B) Temperature changes plotted

against the Cu3(PO4)2@PDA concentrations. Inset shows the linear portion of the curve. (C) Temperature changes recorded in a 500 M J

Cu3(PO4)2@PDA solution with irradiation at


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808 nm for 10 min followed by no irradiation for 18 min. (D) The linear relationship between the cooling time and the negative natural logarithm of the driving force temperature ( ln ).

To evaluate the photothermal property of the Cu3(PO4)2@PDA nanosheets, a series of solutions comprising Cu3(PO4)2@PDA from 30 to 500 M J

were added

into centrifuge tubes, which were then irradiated with an 808 nm laser for 10 min (power density = 0.71 W/cm2). Figure 4A shows that the solution temperature increased with the amount of Cu3(PO4)2@PDA, while the blank did not produce any noticeable temperature change. Specifically, temperature increases of 20.2 and 42.2 °C were observed in 30 and 500 M J

Cu3(PO4)2@PDA solutions, respectively.

These values are much greater than those of the corresponding Cu3(PO4)2 solutions, which at 500 M J

produced a temperature change of 17.8 °C, and at 30 M J

a

temperature rise of only 7.8 °C (cf. Figure S1A in the Supporting Information). These results indicate that the PDA coating increases the photothermal conversion efficiency (PCE). In Figure 4B the temperature increases sharply and linearly with low Cu3(PO4)2@PDA concentrations (see also the inset) and gradually reaches the steady state at 100 M J

Cu3(PO4)2@PDA and beyond. The strong photothermal effect of

Cu3(PO4)2@PDA is the reason why an inexpensive and portable thermometer can be used to detect the temperature change. As detailed in the Supporting Information, the PCE ()) values of Cu3(PO4)2 and Cu3(PO4)2@PDA were deduced to be 22% and 45.2%, respectively. The ultrathin PDA coating resulted in a PCE enhancement by more than two fold. Such a PCE compares favorably to those of other photothermal agents containing PDA, which include PDA-coated Fe3O4 particles (13.1%),40 PDA/Au hollow superparticles (38%),41 and dopamine-melanin colloidal nanospheres (40%)42 and is considerably greater than those of Cu-based photothermal agents, such as the flower-like copper phosphate (41%),24 copper selenide nanocrystals (22%),43 and copper sulfide nanocrystals (16.3%).44 Other than the high PCE inherent in Cu3(PO4)2, the more efficient light absorption by the flat nanosheets is also an


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important contributor. Besides, the good dispersion of Cu3(PO4)2@PDA also significantly improved PCE due to the increase of the effective surface area.

Figure 5. (A) Linear regression of temperature changes (solid squares) plotted against different CRP concentrations (0.42, 0.83, 1.6, 4.2, 8.3, and 16 pM). The inset shows a wider range of concentrations and the simulated Langmuir isotherm. Error bars are RSD values (n=5). (B) The stability of the Cu3(PO4)2@PDA nanosheets as shown by incremental detections of 8.3 pM CRP over a span of 21 days. Error bars are RSD values (n = 5).

We explored the use of antibody-coated Cu3(PO4)2@PDA nanosheets for enhanced detection of protein biomarkers. Quantification of the CRP samples was realized by measuring the heat converted from the laser energy absorbed by the Cu3(PO4)2@PDA-Ab2 nanosheets. The changes increased linearly from 0.42 to 16 pM (1 pM = 0.12 ng/mL) and leveled off beyond 16 pM (inset). The Langmuir isotherm (red curve in the inset) fits the experimental data well in the linear range but deviates from the data at higher CRP concentrations. This trend can be rationalized as follows. In 1:1 antibody-antigen interaction, the Langmuir isotherm, if obeyed, is generally used to fit the responses collected from an antibody-covered sensor produced by conjugate formations from different analyte (antigen) concentrations. This situation is different from the present sandwich-type immunosensing wherein the responses are generated by attachments of Ab2 molecules from a solution of fixed Ab2 concentration. As we45 and others46,47 have shown in the uses of surface plasmon resonance to measure a single analyte concentration at sensor surfaces covered with different antibody densities, the following equation48 should be valid for the steady state reached after attachment of the Cu3(PO4)2@PDA-Ab2 nanosheets:


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= where

[

[

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2]

(1)

2] +

[Ab2]

is

the

concentration

associated

with

a

given

amount

of

Cu3(PO4)2@PDA-Ab2 nanosheets used for the detection, KD is the dissociation equilibrium constant between CPR and the Cu3(PO4)2@PDA-Ab2 nanosheets, and Tmax is the maximal temperature change for a given amount of CRP captured by Ab1. The amount of CRP captured is proportional to the CRP concentration in the sample: (2)

=

where m is a proportionality constant. Combining eqs. 1 and 2 yields: =

[ [

2]

2] +

[

]=

(3)

As the same amount of Cu3(PO4)2@PDA-Ab2 nanosheets was used to collect the data shown in Figure 5A, all the parameters except for [CRP] in eq. 3 are constant and can be represented by the proportionality constant m’. The linear regression (red line) corresponds to XTm = 0.12 [CRP] + 0.37 and the error bars (RSDs) range from 15 to 22%. The small but non-zero intercept could be due to a small exchange of the heat with the ambient and the variation of the nanosheets in size, the latter of which also contributes to the relatively large RSDs. Using this equation and repeated measurements of a CRP-free solution, the detection limit was estimated to be 0.11 pM. The remarkably low detection limit is resulted from the improvement of the detection sensitivity by the following two factors. The first is that conjugation of one CRP with a single Ab2 molecule allows the attachment of a much larger Cu3(PO4)2@PDA nanosheet to the titer well, which produces sizeable heat owing to the nanosheet’s high PCE and flat surface. The second factor that contributes to the enhanced sensitivity is the numerous Ab2 molecules coated onto a single Cu3(PO4)2@PDA nanosheet. The densely populated Ab2 molecules on a single nanosheet favors the formation of the CRP-Ab2 conjugates at the well surface. Indeed, the simulated Langmuir isotherm (red curve in the inset of Figure 5A) yielded an apparent dissociation equilibrium constant of 12.5 pM, which is almost two orders of magnitude smaller than the actual KD between CRP and the Ab2 we used (1.18 nM 49). ACS Paragon Plus Environment

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Both factors help us attain a detection level that is lower than those reported in the literature based on photothermal measurements.22,23 Because of the relatively large dimension of the nanosheet (67.3 ± 2.3 nm), the nanosheet adsorbed onto the titer well sterically hinders the attachment of additional nanosheets. This is the reason behind the deviation of the responses at higher CRP concentrations from the Langmuir isotherm (cf. inset of Figure 5A). As mentioned above, an attractive feature of “nanozymes” is the obviation of an enzyme for signal amplification. This feature helps ensure that the CRP-Ab2 conjugates preformed at the well surface are not dissociated during temperature elevation caused by the photothermal conversion. Moreover, it also contributes to the chemical and biological stabilities we observed for the Cu3(PO4)2@PDA-Ab2 nanosheets. As shown in Figure 5B, the Cu3(PO4)2@PDA-Ab2 nanosheets produced highly comparable assay results during a span of 21 days even after frequent cool-warm cycles of a stock solution stored at 4 °C. The reproducibility of our method was assessed with five assays of 8.3 pM CRP under identical condition (Fig S2A), and the relative standard deviation was calculated to be 2.3%. To verify the absence of non-specific binding, we tested four other antigens, carcinoembryonic antigen, prostate-specific antigen,

-fetoprotein, and cardiac troponin-I. The much smaller

temperature changes (Figure S2B) indicate that the immunosensor is highly specific. To evaluate the reliability for detection of CRP in human serum, our photothermal immunoassay results of five different serum samples nanosheets were compared to those measured with immunoturbidimetry at the hospital (Shanghai Jiao Tong University Medicine School). As shown in Table 1, the relative differences between the two assays are all less than 10%. We also performed a student’s t test (paired t test)50 and obtained a t value of 2.122. This value is smaller than ttable at the 99% confidence level (3.747), suggesting that the differences in Table 1 are statistically insignificant. Therefore, our photothermal immunoassay is accurate and immune to interferences present in complex biological media. Finally we note that the cost of our assay (~$0.5/sample) is only a small fraction of that at the hospital


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($4.7/sample). In practice the cost can be further decreased as more automated analyses can be performed on many samples.

Table1. Assay results of the photothermal immunoassay and immunoturbidimetry Photothermal assay

Immunoturbidimetry

Relative

Sample (Mg/mL)

-M J

/

differences

Healthy donor 1

0.190 ± 0.05

0.210

9.52%

Healthy donor 2

0.590 ± 0.07

0.630

6.35%

Healthy donor 3

1.44 ± 0.08

1.34

7.46%

Patient 1

3.86 ± 0.12

3.63

6.34%

Patient 2

5.22 ± 0.25

5.67

7.94%

CONCLUSIONS We have synthesized polydopamine-encapsulating Cu3(PO4)2@PDA nanosheets and coated them with an antibody for sensitive immunoassay of the C-reaction protein. The flat surface of these nanosheets facilitates greater light absorption and attachment of a large number of antibodies, affording high sensitivity for photothermal detection of protein biomarkers in the sandwich assay format. Consequently, detection of low pM concentrations of proteins can be achieved without the use of antibody-conjugated enzymes for signal amplification. The nanosheet-based “nanozyme” is stable and cost-effective. We have shown that the Langmuir isotherm is also applicable to the sandwich assay at low protein concentrations and attributed the deviation of experimental data from the Langmuir isotherm at high protein concentrations to steric hindrance imposed by the flat nanosheets. Our experimental setup is simple and easy to implement and the analytical “figures of merit” compare favorably to those of conventional methods such as immunoturbidimetry and ELISA commonly employed in clinical and immunological laboratories.


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ASSOCIATED CONTENT Supporting Information Supporting Information Available: Photothermal property of the Cu3(PO4)2 nanosheets, reproducibility and specificity of the immunosensor, and calculation of photothermal conversion efficiency. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] ORCID Feimeng Zhou: 0000-0002-2568-765X

ACKNOWLEDGES The authors would like to thank the start-up provided by The University of Jinan, the financial support from the Natural Science Foundation of China (No. 21245007 and 81000976), and the Natural Science Foundation of Shandong Province (No. ZR2017MB017). F. Zhou and X. W. also thank a 2011 Collaborative and Innovative Grant from Hunan Province of China.


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REFERENCES (1) Porstmann, T.; Kiessig, S. T. Enzyme immunoassay techniques an overview. J. Immunol. Methods, 1992, 150, )N( 9 (2) Vashist, S. K.; Luong, J. H. T. In Handbook of Immunoassay Technologies; Vashist, S. K., Luong, J. H. T., Eds.; Academic Press: 2018, pp B?N (?9 (3) Miao, L.; Zhu, C.; Jiao, L.; Li, H.; Du, D.; Lin, Y.; Wei, Q. Smart Drug Delivery System-Inspired Enzyme-Linked Immunosorbent Assay Based on Fluorescence Resonance Energy Transfer and Allochroic Effect Induced Dual-Modal Colorimetric and Fluorescent Detection. Anal. Chem., 2018, 90, B?=N B

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