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Sensitive and specific detection of L-lactate using an AIE-active fluorophore Zhiling Zhang, Ryan T. K. Kwok, Yong Yu, Ben Zhong Tang, and Ka Ming Ng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10178 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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ACS Applied Materials & Interfaces

Sensitive and specific detection of L-lactate using an AIE-active fluorophore Zhiling Zhang,a Ryan T. K. Kwok,b Yong Yu,a Ben Zhong Tangb and Ka Ming Ng* a a. Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. b. Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. KEYWORDS: Aggregation-induced emission, fluorometry, L-lactate detection, L-lactate oxidase, biomedical diagnostics.

ABSTRACT: L-lactate is a vital biomarker for many diseases and physiological fatigue. An AIE-active fluorophore (TPE-HPro) is combined with L-lactate oxidase (LOx) to determine Llactate in aqueous fluid. The assay shows excellent sensitivity and anti-interference performance with a limit of detection (LOD) of 5.5 µM. In addition, sensitive detection of L-lactate is achieved even in a protein-rich environment. It is proposed that quantification of L-lactate be

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performed at 20 min or 60 min in the current method. These characteristics endow the fluorometric assay with great potential for biomedical diagnostics.

L-lactate, produced by anaerobic respiration of glucose, is a key metabolite for human health assessment. In aerobic respiration, glucose and oxygen react to supply the energy required for human metabolism. When the supply of oxygen is inadequate during high energy demand, anaerobic respiration comes into play, leading to L-lactate production. The L-lactate is produced in all tissues such as skeletal muscle and brain and is cleared mainly by liver and kidney. The balance of production and clearance keeps its level in a normal range to prevent an excessive drop in pH and cell acidosis.1 Previous studies indicate that an abnormal accumulation of Llactate is one of the most important biomarkers for a variety of physiological disorders including hyperlactatemia, cardiogenic shocks, respiratory failure, sepsis, systemic disorders, tissue hypoxia, liver disease and renal failure.1 Therefore, monitoring the L-lactate level of those patients can help assess the severity of diseases and effectiveness of treatments. In addition, as Llactate production is proportional to the extent of physical activity, it serves as an indicator for assessing physiological fatigue of athletes, soldiers and manual laborers.2 The detection of L-lactate is commonly carried out utilizing blood as the sample (which has a typical L-lactate concentration of 1 mM),3 but its invasive nature is known to be a drawback. On the contrary, the use of other body fluids such as sweat and saliva (which have a typical L-lactate concentration of 20 mM and 0.3 mM, respectively),3-4 is gaining more attention due to the noninvasive sampling methods. A number of techniques such as gas chromatography (GC) and highperformance-liquid chromatography (HPLC) have already been employed for L-lactate


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analysis.5-6 However, time-consuming sample pretreatment, complicated equipment handling, and high cost limit their applications. Therefore, the development of a simple, specific, and sensitive L-lactate detection system is highly desired. Electrochemical techniques have been frequently employed in L-lactate sensing. For example, Manna and Raj used covalently functionalized reduced graphene oxide with p-nitrophenyl moiety in an electrochemical biosensor.7 L-lactate dehydrogenase (LDH) converted L-lactate into pyruvate with cofactor NAD+ into NADH, which was then oxidized on the modified electrode and generated current response at +40 mV. This low working potential overcame the interference caused by other electro-oxidizable species in the sample. Wang et al. integrated screen-printed electrodes immobilized with LOx on mouth guard and tattoo for detection of human L-lactate in saliva and sweat, respectively.2, 8 In the presence of LOx, L-lactate was converted into hydrogen peroxide (H2O2), which was measured through electrochemical method. These studies realized noninvasive, continuous monitoring of L-lactate in saliva and sweat for the first time. Colorimetric detection is another widely used sensing method. Dai et al. fabricated a colorimetric paper sensor. NADH, produced from LDH-catalyzed oxidation of L-lactate, reduced the preloaded dye and yielded orange color on the paper sensor.9 The LDH was fused with a cellulose-binding domain, enabling efficient enzyme immobilization for improved sensor stability as well as prolonged sensor life. This sensor showed a linear detection range of 0.5-8 mM. Fluorometric assay is widely applied to chemical and biological detection because of its intrinsic advantages such as high sensitivity, simple construction, and anti-interference performance. In comparison, colorimetric methods usually show inferior sensitivity,10 and many electrochemical assays suffer from multi-step electrode modification and bio-fouling.11 To date, plenty of luminescent materials such as fluorescent proteins, organic dyes, organic nanoparticles,


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inorganic nanoparticles and quantum dots are widely used in chemical and biological applications.12

Marquette

and

Blum

reported

a

L-lactate

biosensor

based

on

electrochemiluminescence of luminol (3-aminophthalhydrazide) by means of flow injection analysis.13 The luminol oxidized on a glassy carbon electrode at +425 mV reacted with H2O2 that generated in the process of LOx-catalyzed L-lactate oxidation. The LOD for L-lactate in this study was as low as 30 pmol with collagen membrane as enzymatic support. The employment of collagen membrane successfully improved the sensitivity, but the fragility of this support hindered its routine use and required further modification.13 Conventional luminophores, however, often suffer from aggregation-caused quenching (ACQ) problem that they show reduced emission in their aggregated state. Opposite to ACQ, aggregation-induced emission (AIE) luminophores emit intensely in an aggregated form, such as those aggregates in solutions and in solid state, thus enabling the development of “turn-on” biosensors by controlling the aggregation.14 In the past decades, researchers have made great efforts to develop various AIE luminogens (AIEgens) and use them for diverse chemo/biodetection.15-20 Recently, Song et al. designed an AIE-active “turn-on” bioprobe for H2O2 quantification.21 The probe employs tetraphenylethylene (TPE) as the core structure, whose emission at around 500 nm is normally weakened by an imine group via a photo-induced electron transfer process either in solution or aggregate. When H2O2 oxidizes the phenylboronic pinacol ester on TPEHPro, a p-quinone methide is released and the remaining product emits bright fluorescence at around 530 nm; that is, it is turned on. In the present work, this probe (TPE-HPro) is combined with LOx for determining the concentration of L-lactate in aqueous fluid. LOx catalyzes the oxidation of L-lactate with the generation of H2O2, which is then quantified by TPE-HPro


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through fluorometry (Scheme 1). The sensing system shows superior sensitivity and antiinterference ability, and still exhibits excellent performance under protein-rich environment.

Scheme 1. Schematic illustration of L-lactate sensing mechanisms. TPE-HPro was synthesized according to the synthetic route reported in our previous article.21 In acetonitrile/water mixtures (1:9 v/v), TPE-HPro has an excitation wavelength of 373 nm and an emission wavelength of 530 nm. The reaction was carried out at 37 °C, the optimum temperature for enzymatic catalysis. A systematic study of the influence of sensing conditions on the detection performance was conducted, including pH, TPE-HPro concentration, and LOx activity. It is believed that both the oxidation of phenylboronic pinacol ester and the enzyme activity are strongly influenced by pH.21 I was the fluorescence intensity of the solution measured in the presence of L-lactate and I0 was the fluorescence intensity of the solution measured without Llactate. As shown in Figure 1A, the relative fluorescence intensity (I/I0) achieved the maximum at pH 11 with a L-lactate concentration of 200 µM, and similar results were obtained at various


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L-lactate concentrations (Figure S1, Supporting Information). When pH < 10, the oxidation of phenylboronic pinacol ester seemed not to proceed completely as reported previously.21 On the other hand, the activity of the enzyme plunged when pH > 11. As an AIE-active fluorophore, the concentration of TPE-HPro in solution plays quite an important role in the detection of L-lactate. The relative fluorescence intensity kept rising as the TPE-HPro concentration was increased up to 60 µM (Figure 1B). However, a concentration higher than that did not raise the signal as expected. This was due to the formation of large aggregates which hinder mass transfer and thus prevent the AIE molecules from reacting with H2O2 thoroughly.22 An adequate amount of LOx is required to ensure complete conversion of L-lactate to H2O2. The results in Figure 1C show that the highest fluorescence intensity was obtained with 1 unit/mL LOx when the enzymatic reaction was carried out at pH 11. Further addition of enzyme actually lowered the fluorescence signal. Previous computational results indicated AIE molecules would dock in the hydrophobic cavity of proteins with the aid of hydrophobic effect, charge neutralization, and hydrogen bonding interactions.23 This interaction between the enzyme protein and TPE-HPro influenced the subsequent oxidation process of TPE-HPro. The effect of oxygen on this enzyme-catalyzed oxidation process was also investigated. No significant difference was shown among the three kinetic curves obtained under different conditions of oxygen supply (Figure S2). The open space and small liquid volume enable rapid and continuous dissolution of oxygen in the buffer, which ensures a sufficient supply of oxygen for the detection. Thus, a pH of 11, 60 µM TPE-HPro and 1 unit/mL LOx were used as the detection conditions in this study. Fluorescence spectra of TPE-HPro were recorded every 6 min after an addition of


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200 µM L-lactate. As presented in Figure 1D, the fluorescence intensity of emission increased gradually with peaks at around 530 nm and the fluorescence reached a plateau around 60 min. An experiment was conducted to verify that the plateau is due to reaction completion rather than enzyme deactivation (Figure S3). 3.5

3.0

A

B

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C

Time (min) 3.0 60 2.5 0 2.0

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1.5

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0.5

1.0

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2.0

LOx (unit/ml)

2.5

3.0

450

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Figure 1. Plot of relative fluorescence intensity at 530 nm versus (A) pH, (B) TPE-HPro concentration and (C) LOx activity. (D) Time-dependent fluorescence spectra of TPE-HPro. Solution: acetonitrile/water mixtures (1:9 v/v); excitation wavelength: 373 nm; pH 11; [TPE-


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HPro]: 60 µM; LOx: 1 unit/mL; temperature: 37 °C; incubation time: 60 min; [L-lactate]: 200 µM. The relative fluorescence intensity at 60 min incubation time versus L-lactate concentrations is plotted in Figure 2. The data were obtained with 3 replicates from aqueous standard solutions containing from 5 × 10-6 to 5 × 10-4 M of L-lactate in the working buffer. As can be seen, a linear relationship was found ranging from 0 µM to 200 µM with a correlation coefficient (R2) of 0.999. When the concentration of L-lactate is above 200 µM, the sample for quantification needs to be diluted until its concentration falls in the linear range. Moreover, the LOD, defined as three-fold standard deviation of the blank (3σ), was 5.5 µM. 4.0

4.0

A

B

3.5

y = 0.00985x+0.96108 R2 = 0.999

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0.5 0

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[Lactate] (µM)

500

0

40

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[Lactate] (µM)

Figure 2. (A) Plot of relative fluorescence intensity at 530 nm versus L-lactate concentration. Solution: acetonitrile/water mixtures (1:9 v/v); excitation wavelength: 373 nm; pH 11; [TPEHPro]: 60 µM; LOx: 1 unit/mL; temperature: 37 °C; incubation time: 60 min; [Lactate]: 0 µM – 500 µM (B) Linear calibration curve of L-lactate assay under the same conditions. [Lactate]: 0 µM – 200 µM.


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In addition to sensitivity, selectivity as another critical parameter for sensing techniques was also evaluated in this work. The most common interfering species in body fluids include urea, pyruvate, glucose, cysteine, citrate and ascorbate.24 The fluorescence responses of this sensing system to these potentially interfering substances in body fluids were recorded and shown in Figure 3A. The results showed a relative activity of 100% for L-lactate and ≤ 1.4% for all other substances. Thus, the system can effectively avoid the interference from all of these substances. As mentioned above, high concentration proteins can interact with TPE-HPro and thus influence the downstream reaction. Therefore, it is necessary to investigate the performance of this sensing system in a protein-rich environment. Herein, 0.1 mg/mL albumin was added in the buffer to mimic the protein rich environment such as saliva.25 As depicted in Figure 3B, the new standard curve still exhibits a linear dependence on L-lactate concentration in the range of 0 µM – 200 µM and a LOD of 7.3 µM. This affirmed the good performance of this assay in the presence of high concentration proteins. 4.0

Ure Pyr

Not Detected

A

B

3.5 y = 0.00843x+1.15963 R2 = 0.999

Not Detected

Lac

3.0 2.5

Glu Not Detected 2.0

I / I0

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0

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Figure 3. (A) Fluorescence responses to potentially interfering substances. [Urea]: 5000 µM; [Pyruvate]: 150 µM; [Lactate]: 200 µM; [Glucose]: 100 µM; [Cysteine]: 1 µM; [Citrate]: 100


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µM; [Ascorbate]: 10 µM. (B) Linear calibration curve of L-lactate assay incubated with 0.1 mg/mL albumin. [Lactate]: 0 µM – 200 µM. Solution: acetonitrile/water mixtures (1:9 v/v); excitation wavelength: 373 nm; emission wavelength: 530 nm; pH 11; [TPE-HPro]: 60 µM; LOx: 1 unit/mL; temperature: 37 °C; incubation time: 60 min. Several fluorescent L-lactate sensors have been reported and the analytical characteristics are listed in Table 1 for a comparison. It is shown that the assay developed in this paper makes further improvements in sensitivity and selectivity over other results. Table 1. Analytical characteristics of fluorescent L-lactate sensor

Enzyme

Luminophore

Linearity

LOD

Selectivity

LOx

TPE-HPro

0 - 200 µM

5.5 µM

Insignificant effect from This paper urea, pyruvate, glucose, cysteine, citrate, ascorbate and protein

LOx

Silole 1

0 - 40 µM

9.2 µM

Insignificant effect from 26 saccharides, amino acids, and ascorbic acid

LDH

Nile-Blue0.05 - 10 mM functionalized CdTe quantum dots hydrogel

50 µM

Insignificant effect from 27 common metal ions, amino acids and other small molecules

LDH

NADH

20 µM

Not reported

0.06 - 1 mM

Reference

28

The rapid determination of L-lactate is important for point-of-care diagnostics. The use of fluorescence signals at 20 min, instead of 60 min, after the addition of L-lactate was also considered (Figure S4). The relative fluorescence intensities were plotted versus L-lactate concentrations in Figure 4. The curve without (Figure 4A) and with (Figure 4B) the incubation of


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albumin both showed a linear increase with L-lactate concentration ranging from 40 µM to 200 µM. 3.0

3.0

A

B y = 0.00418x+1.2519 2 R = 0.994

2.5

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y = 0.00532x+1.16719 2 R = 0.993

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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


0.5

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Figure 4. Linear calibration curve of L-lactate assay (A) without and (B) with incubation of 0.1 mg/mL albumin. Solution: acetonitrile/water mixtures (1:9 v/v); excitation wavelength: 373 nm; emission wavelength: 530 nm; pH 11; [TPE-HPro]: 60 µM; LOx: 1 unit/mL; temperature: 37 °C; incubation time: 20 min; [Lactate]: 40 µM – 200 µM. These experimental results are in agreement with pseudo-first-order reaction kinetics. For such a reaction, the reaction rate is as follows: ௗ௫ ௗ௧

= ݇(ܽ଴ − ‫ܾ)ݔ‬଴

(1)

Here, t is time, x the consumed substrate (L-lactate) concentration at time t, k the reaction constant, and a0, b0 the initial reactant concentrations (L-lactate and TPE-HPro, respectively).29 Integration with the initial condition x = 0 at t = 0 gives ‫ܽ = ݔ‬଴ (1 − ݁ ି௞௕బ ௧ )

(2)

In a fluorescence detection system, the fluorescence intensity quantitatively depends on dye concentration when other conditions remain the same according to the Parker equation:


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‫ܫ‬௙ = 2.3‫ܫ‬௘௫ ߮ߝ‫ܥܭܮ‬

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(3)

Here, If is fluorescence intensity, Iex intensity of excitation light, ߮ quantum yield of fluorescence, ߝ extinction coefficient of dye, L light path length through solution, K instrumental constant, and C dye concentration.30 In the current sensing system, the concentration of produced dye equals to that of consumed substrate; that is, C = x. Thus, the fluorescence intensity of this system at a given time during reaction is proportional to the initial L-lactate concentration. Therefore, quantification can be achieved using the fluorescence signal at 20 min (or another time point during reaction) and after reaction completion. There are pros and cons. A higher signal to noise ratio can be obtained for L-lactate quantification when the reaction is near completion at 60 min. On the other hand, the quantification at 20 min, while faster, involves more uncertainty in detecting low concentrations of L-lactate. In summary, we have successfully developed a novel assay employing AIE-active TPE-HPro and LOx to detect L-lactate in a fluorescence “turn-on” manner. It is a simple and economical method for L-lactate quantification in aqueous medium. The optimum operating conditions - pH, concentration of TPE-HPro, and LOx activity - have been identified. The fluorescence signal approached a steady value around 60 min. The quantification of L-lactate is highly sensitive, with LOD reaching as low as 5.5 µM. The system shows little response to various potential interfering substances and can still detect L-lactate sensitively in systems with high protein content although minor correction using a new calibration is necessary. These characteristics show that the fluorometric assay developed in this study has great potential for L-lactate detection in body fluids, such as sweat and saliva. Therefore, our future work will be realization of this system in neutral environment and its application in real sample analysis.


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ASSOCIATED CONTENT Supporting Information. The following file is available free of charge. Additional information on the experimental descriptions, plot of relative fluorescence intensity at 530 nm versus L-lactate concentration at pH 7, 10 and 11 (Figure S1), kinetic fluorescence responses under different conditions of oxygen supply (Figure S2), kinetic fluorescence responses with an addition of 200 µM L-lactate at 60 min (Figure S3), kinetic fluorescence responses to different concentrations of L-lactate without (A) and with (B) incubation of 0.1 mg/mL albumin (Figure S4) (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Innovation and Technology Commission (ITC-CNERC14SC01). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work was partially supported by the Innovation and Technology Commission (ITCCNERC14SC01). ABBREVIATIONS LOx, L-lactate oxidase; LOD, limit of detection; GC, gas chromatography; HPLC, highperformance-liquid chromatography; LDH, L-lactate dehydrogenase; NAD+, nicotinamide adenine dinucleotide; NADH, reduced form of nicotinamide-adenine dinucleotide; H2O2, hydrogen peroxide; ACQ, aggregation-induced quenching; AIE, aggregation-induced emission; AIEgens, AIE luminogens; TPE, tetraphenylethylene. REFERENCES (1) Rassaei, L.; Olthuis, W.; Tsujimura, S.; Sudhölter, E. J.; van den Berg, A. Lactate Biosensors: Current Status and Outlook. Anal. Bioanal. Chem. 2014, 406 (1), 123-137. (2) Jia, W.; Bandodkar, A. J.; Valdés-Ramírez, G.; Windmiller, J. R.; Yang, Z.; Ramírez, J.; Chan, G.; Wang, J. Electrochemical Tattoo Biosensors for Real-Time Noninvasive Lactate Monitoring in Human Perspiration. Anal. Chem. 2013, 85 (14), 6553-6560. (3) Hickey, D. P.; Reid, R. C.; Milton, R. D.; Minteer, S. D. A Self-Powered Amperometric Lactate Biosensor Based on Lactate Oxidase Immobilized in Dimethylferrocene-Modified LPEI. Biosens. Bioelectron. 2016, 77, 26-31. (4) Segura, R.; Javierre, C.; Ventura, J. L. L.; Lizarraga, M. A.; Campos, B.; Garrido, E. A New Approach to the Assessment of Anaerobic Metabolism: Measurement of Lactate in Saliva. Br. J. Sports Med. 1996, 30 (4), 305-309.


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(5) Playne, M. J. Determination of Ethanol, Volatile Fatty-Acids, Lactic and Succinic Acids in Fermentation Liquids by Gas-Chromatography. J. Sci. Food Agric. 1985, 36 (8), 638-644. (6) Milagres, M. P.; Brandao, S. C. C.; Magalhaes, M. A.; Minim, V. P. R.; Minim, L. A. Development and Validation of the High Performance Liquid Chromatography-Ion Exclusion Method for Detection of Lactic Acid in Milk. Food Chem. 2012, 135 (3), 1078-1082. (7) Manna, B.; Raj, C. R. Covalent Functionalization and Electrochemical Tuning of Reduced Graphene Oxide for the Bioelectrocatalytic Sensing of Serum Lactate. J. Mater. Chem. B 2016, 4 (26), 4585-4593. (8) Kim, J.; Valdés-Ramírez, G.; Bandodkar, A. J.; Jia, W. Z.; Martinez, A. G.; Ramírez, J.; Mercier, P.; Wang, J. Non-Invasive Mouthguard Biosensor for Continuous Salivary Monitoring of Metabolites. Analyst 2014, 139 (7), 1632-1636. (9) Dai, G. Y.; Hu, J. L.; Zhao, X. Y.; Wang, P. A Colorimetric Paper Sensor for Lactate Assay Using a Cellulose-Binding Recombinant Enzyme. Sens. Actuators, B 2017, 238, 138-144. (10) Haugland, R. P. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Molecular probes: 2005. (11) Cosnier, S. Biosensors Based on Electropolymerized Films: New Trends. Anal. Bioanal. Chem. 2003, 377 (3), 507-520. (12) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. AggregationInduced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115 (21), 1171811940.


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