Smart Drug Delivery System-Inspired Enzyme-Linked Immunosorbent

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Smart drug delivery system-inspired enzyme-linked immunosorbent assay based on FRET and allochroic effect induced dual-modal colorimetric and fluorescent detection Luyang Miao, Chengzhou Zhu, Lei Jiao, He Li, Dan Du, Yuehe Lin, and Qin Wei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04068 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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Analytical Chemistry

Smart drug delivery system-inspired enzyme-linked immunosorbent assay based on FRET and allochroic effect

induced

dual-modal

colorimetric

and

fluorescent detection Luyang Miaoǁ, Chengzhou Zhuǂ, Lei Jiaoǀ, He Liǀ, ǁ*, Dan Duǂ, Yuehe Linǂ*, Qin Weiǁ ǀ College of Optoelectronics Technology, Chengdu University of Information Technology, Chengdu 610225, China. E-mail: [email protected] (H. Li) ǁ

ǂ

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China. School of Mechanical and Materials Engineering, Washington State University, Pullman,

Washington 99164, United States E-mail: [email protected] (Y. Lin)

ACS Paragon Plus Environment

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KEYWORDS: drug delivery system, enzyme-linked immunosorbent assay, fluorescence resonance energy transfer, allochroic effect, dual-modal detection

ABSTRACT: Numerous analytical techniques have been undertaken for the detection of protein biomarkers due to their extensive and significant applications in clinical diagnosis, whereas there are few strategies to develop dual-readout immunosensors to achieve more accurate results. To the best of our knowledge, inspired by smart drug delivery system (DDS), a novel pHresponsive modified enzyme-linked immunosorbent assay (ELISA) was innovatively developed for the first time, realizing dual-modal colorimetric and fluorescent detection of cardiac troponin I (cTnI). Curcumin (CUR) was elaborately selected as report molecule, which played the same role of drugs in DDS based on the following considerations: 1) CUR can be used as a kind of pH indicator by the inherited allochroic effect induced by basic pH value; 2) the fluorescence of CUR can be quenched by certain nanocarriers as the acceptor due to the occurrence of fluorescence resonance energy transfer (FRET), while recovered by the stimuli of basic pH value, which can produce “signal-on” fluorescence detection. Three-dimensional MoS2 nanoflowers (3D-MoS2 NFs) were employed in immobilizing CUR to constitute nanoprobe for the determination of cTnI by virtue of good biocompatibility, high absorption capacity and fluorescence quench efficiency towards CUR. The proposed DDS inspired ELISA offered dualmodal colorimetric and fluorescent detection of cTnI, thereby meeting the reliable and precise analysis requirements. We believe that the developed dual-readout ELISA will create a new avenue and bring innovative inspirations for biological detections.


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INTRODUCTION The accurate determination of disease biomarkers has become one of the core tasks in the fields of biomedical diagnosis and prognosis.1,

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Tremendous research enthusiasm has been

contributed to explore reliable strategies for the detection of disease biomarkers. Particularly, immunoassay following electrochemical,3 paramagnetic,4 colorimetric,5 fluorescence,6 surface plasmon resonance,7 etc. readout strategies has been endowed as the most promising approach. It should be noted that enzyme-linked immunosorbent assay (ELISA) has so far been the most welcomed quantitative technique. Although ELISA is featured with acceptable specificity and sensitivity, its deficiencies are also conspicuous. Firstly, enzymes as signal enhancer are less stable and vulnerable to the impacts of the activity of enzyme. Secondly, the sensitivity is difficult to reach picogram level and unable to satisfy the detection requirement for the protein analytes with relatively low content in human body (such as the cardiac troponin I (cTnI), its cutoff value is almost zero).8, 9 Thirdly, the signal readout is ascertained only through color change and spectrometer-assisted absorbance to ultimately implement the quantitative analysis of model targets. Furthermore, the dependence for single model readout makes the results of analytical performance uncertain. Therefore there is an urgently desire for the improvement of ELISA to circumvent its shortcomings and obtain the more excellent stability, sensitivity and accuracy. Significant endeavors have been contributed to explore modified ELISA with superior performance for realizing the more sensitive, precise detection of targets. For instance, our group explored a bioinspired strategy to prepare “all-in-one” organic-inorganic hybrid nanoflowers for the sensitive detection of E.coli O157:H7, which brought a universal approach to resolve the bottleneck of conventional ELISA by the improved enzyme activity and stability of the nanoflowers as signal labels.10,11 Besides, to develop enzyme-free based ELISA will be a


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promising alternative approach. Following this strategy, Pd-Ir core-shell nanocubes as peroxidase mimic were used for colorimetric immunoassay of PSA following ELISA model, achieving much more sensitive detection performance compared with conventional ELISA due to the merits of high efficiency and superior stability of Pd-Ir nanocubes.12 However, it is noted that the above-mentioned techniques are all followed single-modal signal readout, which may be susceptible to the external interferences and difficult to meet the requirements of the precise and highly sensitive bioassay. In this case, a dual-model modified ELISA provides a promising alternative for more reliable biological detection. Smart drug delivery systems (DDS) possess the stimulus-responsive feature to various external/internal physical, chemical and biological stimuli, which bring fascinating opportunities in precision medicine.13 Hinted by the core essential of smart DDS, an inventively improved ELISA was proposed, which was based on fluorescence resonance energy transfer (FRET) and allochroic effect induced dual-model colorimetric and fluorescent readout. Herein, cTnI was chosen as the model analyst because cTnI is now considered as the most valuable diagnostic marker for acute myocardial infarction (AMI).14, 15 There is a highly urgent demand for fabricating a reliable, precise and ultrasensitive detection approach for cTnI. Curcumin (CUR) as a natural yellow pigment extracted from ginger root has been used for cancer therapy due to the induced high toxicity.16 CUR was elaborately chosen as report molecules for the first time based on the following considerations: 1) CUR can be used as a kind of pH indicator by the inherited allochroic effect induced by basic pH value;17 2) the fluorescence of CUR can be quenched by certain nanocarriers as the acceptor due to the occurrence of FRET, while recovered by the stimuli of basic pH value, which can produce “signal-on” fluorescence detection. MoS2 nanomaterials showed great promising in biomedical applications, such as cancer therapy,


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biosensing and wound disinfection, by virtue of good biocompatibility, enzyme mimic property and high fluorescence quench efficiency.18, 19 Especially, MoS2 nanomaterials exhibited good loading capacity towards some hydrophobic drugs, eg. CUR, doxorubicin, resveratrol through hydrophobic interaction.20-22 Therefore, three-dimensional MoS2 nanoflowers (3D-MoS2 NFs) were employed to fabricate signal labels by co-immobilizing report antibodies and CUR as signal reporting molecules. Thanks to the hydrophobic interaction, large amounts of CUR could be absorbed in 3D-MoS2 NFs and the fluorescence would be quenched due to the occurrence of FRET from CUR to MoS2. When the stimulus of basic water was put, hydrophobic CUR turned into hydrophilic ion, leading to the break of the hydrophobic interactions with MoS2. As a result, the adsorbed CUR would be released quickly from the surface of 3D-MoS2 NFs, resulting in color change and fluorescence recovery simultaneously, which can afford dual-modal colorimetric and fluorescence detection. EXPERIMENTAL SECTION Chemicals and materials. Sodium molybdate (Na2MoO4⋅2H2O), thiourea (CH4N2S), thioglycolic acid (TGA, 90.0 %), curcumin (CUR) and Bovine serum albumin (BSA, 96-99 %) were bought from Macklin Biochemical Technology Co., Ltd (Shanghai, China). N-ethyl-N’-(3dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were obtained from Aladdin Industrial Corporation (Shanghai, China). The cardiac troponin I (cTnI) and paired antibodies were purchased from Linc-Bio Science Co., Ltd (Shanghai, China). All the other reagents are analytical grade and utilized directly without further treatments. Ultrapure water was used throughout the experiments. Basic water (BW) in this study was NaOH aqueous solution (pH 12.25) except explained specifically.


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Instruments. Scanning electron microscopic (SEM) images were recorded by Quanta FEG250 field emission environmental SEM (FEI, USA) operated at 4 KV. X-ray powder diffraction (XRD) was performed with a D8 advance X-ray diffractometer (Bruker AXS, Germany). Ultraviolet-visible (UV-vis) measurements were carried out by using a UV–2600 spectrometer (Shimazduo, Japanese). The Zeta potential was measured with a Malvern Instrument (Zetasizer Nano-ZS90). The absorption intensities and all fluorescence measurements in the 96-microwell plates were identified by a Spark 20M Multimode Microplate Reader (TECAN, Switzerland). Synthesis of 3D-MoS2 NFs. 3D-MoS2 NFs were prepared according to the reported procedure with some modifications.23 In brief, 3.7 mmol sodium molybdate and 15.8 mmol thiourea were dispersed into 25 mL ultrapure water with ultrasonic treatment to form a homogenous solution. Subsequently, 12 mL HCl (1 M) was pipetted to the solution. After a few minutes of bath sonication, the obtained solution was transferred into a 50 mL-volume Teflon-lined stainlesssteel autoclave and heated at 220 °C for 22 h. When the hydrothermal reaction was completed, the resultant 3D-MoS2 NFs were centrifuged and washed three times with ultrapure water. Synthesis of MoS2-Ab2-CUR. 1 mg⋅mL⁻¹ 3D-MoS2 NFs were dispersed in 1 mL of ultrapure water with ultrasonic treatment. 12 mg⋅mL⁻¹ thioglycolic acid (TGA) was added into the MoS2 suspension with stirring for 90 min to obtain carboxyl functional 3D-MoS2 NF (C-MoS2 NF).24 Subsequently, the solution was centrifuged and washed three times and re-dispersed in 2 mL of PBS (0.1 M, pH 7.4). Next, the C-MoS2 NF were activated by adding an aqueous mixture of 2 mg mL⁻¹ EDC and 4 mg mL⁻¹ NHS with gently shaking for 30 min at 25 °C. After centrifugation and washing for several times, 1 µg⋅mL⁻¹ cTnI-Ab2 was dropped into the suspension and placed


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at an oscillator at 4 °C for at least 6 h to covalently immobilize Ab2 onto the surface of MoS2 NFs. The mixture was centrifuged for three times to remove any free Ab2 that did not combine with MoS2 NF, and then re-dispersed in 2 mL of PBS. Thereafter, CUR ethanol solution (25 mM, 200 µL) was pipetted into the obtained MoS2-Ab2 suspension with gentle shaking for 2 h to absorb CUR onto the MoS2 surface. Eventually, MoS2-Ab2-CUR conjugates were gathered by centrifuging several times with ethanol and water to remove redundant CUR and re-dispersed in 2 mL of ultrapure water, and the as-prepared suspension was stored at 4 °C before use. Procedure of dual-modal colorimetric and fluorescence immunoassay. In brief, a series of 50 µL of 1 µg⋅mL⁻¹ of monoclonal primary antibody (Ab1) was dropped onto the 96-microwell plate and incubated overnight at 4 °C. After each well was washed for three times with PBS, 50 µL of 1 % BSA was added to the tested wells for blocking the nonspecific activity sites. Subsequently, the incubation process was performed at 37 °C for 1 h and free BSA was removed with PBS. Then, 50 µL of cTnI with different concentrations were pipetted into the 96-microwell plate and incubated at 37 °C for 1 h resulting in specific binding between antigens and antibodies. Similarly, each well was added by MoS2-Ab2-CUR (0.5 mg⋅mL⁻¹, 50 µL) after washed for three times with PBS and incubated at 37 °C for 1 h. As followed, after washing three times with ultrapure water, 100 µL of release reagents (basic water, BW) was put into each well to cause the rapid release of CUR. In order to produce uniform color, the plate was placed at an oscillator for 5 min gently shaking. Finally, the absorbance intensities were recorded via TECAN multimode microplate reader at the maximum absorption wavelength of 468 nm. The fluorescence intensities were following measured with the specific parameters: the excitation and emission slit are 20 nm; the excitation wavelengths of the fluorophores are set at 490 nm; the emission spectra range is 550 to 700 nm.


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Immunoassay on the conventional ELISA. The similiar detailed procedures were utilized for the conventional 96-microwell plates. The condition of incubating capture antibodies was same as the DDS-ELISA. After removal of the unbound antibodies, 50 µL of 1 % blocking solution was added into each well and incubated at 37 °C for 1 h. Then, the plate was washed three times with PBS and 50 µL of cTnI antigens with various concentrations were dropped on the well to incubate at 37 °C for 1 h. Similarly, the plate was washed three times with PBS and then the enzyme-labelled antibodies (100 µL) were added to each well to incubate at 37 °C for 1 h. Subsequently, 100 µL of TMB substrate solution was added after washing three times with water, and incubated at room temperature for 10 min. Finally, the stop buffer was dropped onto the plate, and next the absorbance value was recorded by the TECAN multimode microplate reader at 656 nm. After fitting the calibration curve (respectively from the conventional ELISA and DDS-ELISA), the human serum samples with known cTnI concentration was added to two types of sensor system, following the sensor established and the measured signal collected, the antigens concentration in human serum was respectively calculated by the linear relation equation and fitted to achieve comparison between the conventional ELISA and the DDS-ELISA. RESULTS AND DISCUSSION The fabrication process of signal nanotags was demonstrated in Scheme 1, in which the anchor of the second antibody of cTnI and CUR as signal reporting “drug” on the surface of 3DMoS2 NFs was achieved simultaneously. To immobilize the second antibody of cTnI, MoS2 NF were firstly carboxyl functionalized via the formation of disulfide bond between free sulfur atoms of MoS2 and sulfhydryl of thioglycolic acid. The obtained carboxyl functionalized MoS2


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Scheme 1. Diagram of Drug delivery system inspired enzyme-linked immunosorbent assay (DDS-ELISA) for the detection of cTnI in 96-microwell plates.

NFs (C-MoS2 NFs) were then used for bonding the second antibody of cTnI through EDC and NHS induced carbodiimide coupling. Thereafter, abundant CUR could absorb onto the surface of MoS2 petals by means of hydrophobic interactions. The resultant MoS2-Ab2-CUR conjugates were then used as signal nanotags to fabricate the sandwich immunosensor for cTnI detection. Upon the addition of basic solution as stimulus into well, the loaded CUR would convert into hydrophilic salt and speedily separate from MoS2 NFs, resulting in the colour change distinctly. At the same time, after the CUR were released from signal nanolabels, as expected, the quenched fluorescence signal of CUR would recover due to the break of FRET from MoS2 NFs. Thus, similar to smart responsive DDS, with the trigger of basic solution, the proposed DDS-ELISA could present dual-modal colorimetric and fluorescence detection of cTnI. Characterization of 3D-MoS2 NF and MoS2-Ab2-CUR. As revealed by the SEM images of Figure 1B and 1C, the as-synthesized MoS2 nanostructures exhibited a well-defined morphology


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of three-dimensional hierarchical nanoflowers with abundant and dense nanopetals, which have a similar feature with marigold in Figure 1A. The XRD analysis confirmed that 3D-MoS2 NFs has the crystal structure of 2H-MoS2. As indicated in Figure 1D, three diffraction peaks at 14.0° 33.5° and 57.3° correspond to the feature peaks of MoS2 (JCPDS 37-1492). The successful carboxyl functionalization of 3D-MoS2 NFs was identified by the change of zeta potential from 21 mV to -26.8 mV. As shown in Figure 1E, the negligible colour change was observed when pure MoS2 and MoS2-Ab2 were dispersed separately in neutral water (NW) and basic water (BW, pH 12.25). However, once triggered by BW, obvious allochroic effect occurred to MoS2-Ab2CUR, indicating the successful loading of CUR on MoS2-Ab2. This phenomenon was attributed to the fact that phenolic hydroxyl group of hydrophobic CUR loaded in MoS2 petal transformed into hydrophilic CUR anions induced by hydroxyl ions. As a result, hydrophilic CUR anions removed from the surface of MoS2 entered into an aqueous solution and formed the reddish brown solution accordingly. As expected, no absorption peak for MoS2 was observed from the UV-vis spectra in Figure 1F. After covalent binding with Ab2, an obvious absorption peak of antibody appeared at 280 nm. A new peak of CUR with very weak absorption intensity appeared at around 428 nm for MoS2-Ab2-CUR, revealing CUR was indeed absorbed onto MoS2. Upon addition of BW, the colour change was found accompanied by the emergence of significant absorbance at the peak of 468 nm with a red-shift, which was due to the conversion of hydrophobic CUR into water-soluble CUR anions.


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Figure 1. Photograph of marigold flowers (A). SEM (B, C) images of 3D-MoS2 NF. XRD pattern of 3D-MoS2 NFs (D). The photographs of MoS2, MoS2-Ab2 and MoS2-Ab2-CUR dispersed in NW and BW, respectively. (E). UV-vis spectra of aqueous solutions including MoS2, MoS2-Ab2, MoS2-Ab2-CUR and MoS2-Ab2-CUR treated with BW (F).

The absorption capacity of 3D-MoS2 NFs to CUR is a critical parameter for DDS-ELISA because dual-modal colorimetric and fluorescence signal was originated from the detached CUR anions from MoS2-Ab2-CUR. Firstly, with the addition of BW (pH 12.25) to CUR ethanol solution, a standard calibration curve of CUR was built with the linear concentration range of 0.01 mM to 2 mM (Figure 2). With the aid of the linear regression equation of CUR, the absorption capacity of CUR is calculated to be 624 mg/g. The impressively high loading capacity of CUR will make great contribution to improving the sensitivity of DDS-ELISA.


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Figure 2. The standard calibration curve of CUR with the linear concentration range of 0.01 mM to 2 mM

The quenching ability of 3D-MoS2 NFs. The quenching ability of 3D-MoS2 NFs was investigated and shown in Figure 3. The maximum emission peak was located at 545 nm for CUR ethanol solution (ES) when excited at 365 nm. A sharp decrease of fluorescence intensity at 545 nm was observed after CUR was tightly anchored on the surface of MoS2 NFs, and the quenching efficiency was determined to be as high as 95.7 %. This observation revealed that MoS2 NFs owned high FRET capacity towards CUR. Upon treatment with BW, the quenched fluorescence emission of CUR was recovered with a red shift from 545 nm to 612 nm.


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Figure 3. Fluorescence emission spectra of CUR ES (a), MoS2-CUR (b) and MoS2-CUR treated with BW (c) excited at 490 nm. The photographs were shot under the UV irradiation at 365 nm, the concentration of the above three is 0.0238 mg⋅mL⁻¹.

Optimization of the detection condition. The pH value of BW plays a critical role in the colorimetric and fluorescent assay. In consideration of the hydrophobic nature of CUR, the pH value of BW was investigated in alkaline range (Figure 4). It is found that the maximum signal response of both absorbance and fluorescence intensity presented at pH=12.25, which was adopted as optimized one in this work. Moreover, the time of CUR detaching from the MoS2 surface is also an important parameter for fluorescence recovery. When BW was pipetted into the MoS2-Ab2-CUR suspension, the color change was observed immediately and the corresponding absorbance and fluorescence intensity would attain the peak position, indicating that the release process of CUR was very rapid. Consequently, the 96-microwell plate was oscillated gently for 30 seconds for measurements after the trigger of BW.


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Figure 4. The optimization of pH value of BW was conducted by colorimetric (A) and fluorescent (B) assay.

Analytical performance on dual-modal colorimetric and fluorescent detection of cTnI. Under the optimal conditions, the developed DDS-ELISA afforded dual-modal immunoassay of cTnI, achieving the same linear detection range of 0.0005 to 5 ng⋅mL⁻¹ for both colorimetric and fluorescent readout. For colorimetric detection (Figure 5A and B), with the increase of cTnI level, the color of detection solution become darken gradually and the absorbance increased accordingly with semi-quantitative detection limit of 0.5 pg⋅mL⁻¹ via the naked-eye readout. The linear relationship was obtained and the regression equation was Y=1.2780+0.2737*X (R=0.985), and the low detection limit was achieved as low as 0.081 pg⋅mL⁻¹ (S/N=3). What is the most significant is that DDS-ELISA enables a very sensitive naked-eye readout even the concentration of cTnI is as low as 1 pg.mL-1, which is well fitted with for clinical detection of cTnI. Meanwhile, for fluorescence detection (Figure 5C and D), the restored emission fluorescence intensities at 612 nm increased progressively with the increase of cTnI. The linear response of fluorescence intensity with varying concentrations of cTnI and its logarithm values were determined accordingly. The calibration curve was Y=500.3203+82.9488*X (R=0.994) with the low detection limit of 0.074 pg⋅mL⁻¹ (S/N=3). Compared with other techniques for detecting cTnI,


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DDS-ELISA possessed a unique dual signal readout merit and exhibited a more reliable sensitive performance (Table 1).

Figure 5. Calibration curve based on the absorbance for cTnI (A) and log values of cTnI (B). Fluorescence intensity curves acquired by pipetting to BW at MoS2-Ab2-CUR/cTnI/Ab1/plates after incubation with (a) 0.0005, (b) 0.001, (c) 0.005, (d) 0.01, (e) 0.05, (f) 0.1, (g) 1, (h) 5 ng⋅mL⁻¹ cTnI (C). Calibration plots of the fluorescence intensity of different concentrations of cTnI and its log values (D). Error bar = RSD (n = 5).


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Table1. The comparison of DDS-ELISA with other analytical techniques Testing Platform Colorimetric Fluorescence Electrochemical Photo-electrochemical SPR pH meter DDS-ELISA

Linear range 0.1-500 ng⋅mL⁻¹ 0.005-10 ng⋅mL⁻¹ 0.001-0.3 ng⋅mL⁻¹ 0.005-20 ng⋅mL⁻¹ 0.15-660 ng⋅mL⁻¹ 0.01-100 ng⋅mL⁻¹ 0.0005-5 ng⋅mL⁻¹

Detection Limit 0.20 ng⋅mL⁻¹ 2 pg⋅mL⁻¹ 1 pg⋅mL⁻¹ 1.756 pg⋅mL⁻¹ 0.068 ng⋅mL⁻¹ 10 pg⋅mL⁻¹ 0.081 or 0.074 pg⋅mL⁻¹

Reference 25 26 27 28 29 30 This work

Reproducibility and specificity. Figure 6 (red and cyaneous bars) shows the specificity of the developed DDS-ELISA by means of colorimetric and fluorescent readout, respectively. Experiments were carried out by selecting 0 ng⋅mL⁻¹ cTnI as control group, 0.1 ng⋅mL⁻¹ alphafetoprotein (AFP), 0.1 ng⋅mL⁻¹ carcinoembryonic antigen (CEA), 0.1 ng⋅mL⁻¹ C-reactive protein (CRP), 0.1 ng⋅mL⁻¹ cTnI, mixture including 0.1 ng⋅mL⁻¹ cTnI, 10 ng⋅mL⁻¹ AFP, 10 ng⋅mL⁻¹ CEA and 10 ng⋅mL⁻¹ CRP as model mixed antigens. The results displayed the absorbance values and fluorescence intensities of other biomarkers were almost negligible except the cTnI and mixture, revealing the satisfactory specificity of DDS-ELISA. The reproducibility of DDS-ELISA was evaluated under the same conditions. As demonstrated in Figure 6 (green and cerulean bars), five groups of immunoassay for 0.1 ng⋅mL⁻¹ cTnI were performed, respectively. It is satisfied that the proposed DDS-ELISA achieved an excellent reproducibility and the relative standard deviation (RSD) was only 1.35 %.


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Figure 6. Absorbance value (magnified two hundred times, 1) and fluorescence intensity (3) after CUR release via adding to pH 12.25 BW to blank, 0.1 ng⋅mL⁻¹ AFP, 0.1 ng⋅mL⁻¹ CEA, 0.1 ng⋅mL⁻¹ CRP, 0.1 ng⋅mL⁻¹ cTnI, mixture including 0.1 ng⋅mL⁻¹ cTnI, 10 ng⋅mL⁻¹ AFP, 10 ng⋅mL⁻¹ CEA and 10 ng⋅mL⁻¹ CRP (1, 3). Absorbance value (magnified two hundred times, 2) and fluorescence intensity (4) of the proposed immunosensor were presented towards the same cTnI concentration of 0.1 ng⋅mL⁻¹.

Conventional ELISA versus DDS-ELISA. The reliability and practical value of the proposed DDS-ELISA was further validated with the conventional ELISA (Figure 7). It can be observed that a satisfactory linear correlation (R2=0.96) between the two sets of analysis results derived from two various techniques. These results indicated that the two approaches did not show any remarkable difference in the quantitative analysis of cTnI. Concerning that DDSELISA have more advantages involving unique dual-modal readout detection, more feasible and sensitive performance over conventional ELISA, DDS-ELISA may have more promising potential for clinical applications beyond the detection of cTnI.


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Figure 7. Correlation between data obtained from the DDS-ELISA (black) and conventional ELISA (red) for human serum sample with cTnI levels ranging from 0.1 ng⋅mL⁻¹ to 1.5 ng⋅mL⁻¹. Error bar = RSD (n = 5).

CONCLUSION In general, an innovative DDS-ELISA with dual colorimetric and fluorescent signal readout has been constructed for sensitive, precise and reliable detection of cTnI. The excellent performance can be attributed to the following reasons: (1) 3D-MoS2 NFs provided an outstanding platform to immobilize report protein and CUR for dual-model signal readout; (2) CUR played a unique and important role in realizing the dual-modal signal readout by the inherited FRET and allochroic effect. Similar to smart responsive DDS, simply triggered by basic water, dual-modal colorimetric and fluorescent detection can be achieved for DDS-ELISA, which can effectively avoid the facing bottlenecks of conventional ELISA. Accordingly, we strongly believe that the developed dual-readout ELISA will create a new avenue and bring innovative inspirations for biological detections.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y.H. Lin). * E-mail: [email protected] (H. Li). ORCID He Li: 0000-0003-3462-1377 Yuehe Lin: 0000-0003-3791-7587 Author Contributions The main idea of this study was derived from Y.L. and H.L. Then L.M., C.Z., L.J., Q.W. and D.D. conducted most of the experiments. L.M., C.Z., Q.W., Y.L. and H.L. drafted or revised the paper. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The authors would like to thank the Natural Science Foundation of China (No. 21245007 and 81000976) and the Natural Science Foundation of Shandong (No. ZR2017MB017) for the financial support. ABBREVIATIONS DDS, drug delivery system; ELISA, enzyme-linked immunosorbent assay; cTnI, cardiac troponin I; CUR, curcumin; 3D-MoS2 NF, three-dimensional MoS2 nanoflowers; FRET, fluorescence resonance energy transfer; AMI, acute myocardial infarction; SEM, scanning electron microscopic; XRD, X-ray powder diffraction; UV-vis, Ultraviolet-visible; BW, basic water; Ab1, monoclonal primary antibody; Ab2, second antibody; Ag, antigen.


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