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Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognition-induced Multi-DNA Release and Cyclic Enzymatic Amplification Huilei Dong, Hongfei Chen, Juqian Jiang, Hui Zhang, Chenxin Cai, and Qingming Shen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04863 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Analytical Chemistry

Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognition-induced Multi-DNA Release and Cyclic Enzymatic Amplification Huilei Dong,† Hongfei Chen,† Juqian Jiang, † Hui Zhang,*, † Chenxin Cai† and Qingming Shen*,‡ †

Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation

Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, P. R. China. ‡

Key Laboratory for Organic Electronics and Information Displays & Institute of

Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM)), Nanjing University of Posts & Telecommunications, Nanjing 210023, China

*Corresponding authors: Hui Zhang. E-mail: [email protected]. Telephone: (025)8589-1780. Fax: (025)8589-1767 Qingming Shen E-mail: [email protected] Telephone: (025)8586-6827. Fax: (025)8586-6396

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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: Sensitive and specific detection of tumor exosomes is of great significance for early cancer diagnosis. In this paper, we report an aptamer strategy for exosome detection based on aptamer recognition-induced multi-DNA release and cyclic enzymatic amplification. First, we use aptamer-magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells, leading to the release of three kinds of messenger DNAs (mDNAs). After magnetic separation, the released mDNAs hybridized with the probe DNAs immobilized on a gold electrode. Electroactive Ru(NH3)63+ was used as the signal reporter because of its electrostatic attraction to DNA. Subsequent Exo III cyclic digestion caused the electrochemical signal to “turn off”. Since the electrochemical signal reflects the concentration of Ru(NH3)63+ and the concentration of Ru(NH3)63+ is correlated with the mDNA concentration, which is correlated with the exosome concentration, the tumor exosomes can be detected by examining the decrease in the peak current of Ru(NH3)63+. In this paper, the signal was amplified by the numerous mDNAs released from the magnetic bead and the Exo III-assisted mDNA recycling. Under the optimal conditions, a detection limit down to 70 particles/µL was achieved, which is lower than the LODs of most currently available methods. Furthermore, this assay can be used to detect tumor exosomes in complex biological samples, demonstrating potential application in real sample diagnosis.


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INTRODUCTION Exosomes, nanoscale membrane-enclosed extracellular vesicles with diameters of 30-100 nm, are produced by various cells including reticulocytes, leukocytes, neurons, epithelial cells, and tumor cells.1–4 Exosomes carry a considerable amount of molecular information derived from the originating cells, including lipids, proteins and nucleic acids.5–10 Because transfer of these biomolecules from parent cells to recipient cells via exosomes7,9 is thought to contribute to cancer progression and metastasis, exosomes are believed to be potential cancer biomarkers for early cancer diagnosis.11–13 Therefore, it is important to develop analytical techniques for the sensitive and specific detection of tumor exosomes. To date, numerous methods have been used to detect exosomes, such as flow cytometry,14,15 nanoparticle tracking analysis (NTA),16,17 surface plasmon resonance (SPR)18–20 and fluorescence.21 Flow cytometry provides high throughput detection, but is not sensitive for exosomes because of the weak light scattering from particles with diameters less than 100 nm.14 NTA is a powerful technique for exosome quantification, since it allows visualization of individual exosomes in solution, but NTA is primarily designed for zeta-potential determination and/or accurate size distribution.16 Fluorescence is capable of detecting exosomes with high sensitivity, but the approach requires a sufficient number of markers on the exosome surface to achieve a fluorescence emission signal. In addition, these methods require expensive instrumentation. Electrochemical methods have the advantages of low cost and portability, and have been shown to offer high sensitivity in biomolecule detection. Since exosomes carry many biomarkers, antibodies or aptamers, which have good specificity and high binding affinities, can be used to recognize and interact with the biomarkers. By integration with electrochemical labeling, exosomes can be detected directly.22–26 For example, Doldán et al immobilized rabbit antihuman CD9 antibodies on a gold electrode to capture exosomes, which were further captured by mouse antihuman CD9 antibody and horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. Exosome detection was based on the electrochemical reduction of 3,3 ′ ,5,5 ′ -tetramethyl benzidine (TMB).22 Jeong et al. modified a gold electrode with magnetic beads harboring anti-CD63 antibodies to capture exosomes and then further capture HRP-conjugated α-CD63 antibodies, with exosome detection based on the oxidation



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of TMB.23 Recently, Wang et al. reported a nanotetrahedron (NTH)-assisted electrochemical biosensor for direct capture and detection of exosomes. They immobilized DNA NTHs containing the DNA aptamer on the surface of a gold electrode. The aptamer could capture the exosomes, and the ferricyanide-ferrocyanide redox couple was used for signal generation.25 Compared with antibodies, aptamers have several advantages, such as easy and reproducible preparation and modification, and lack of immunogenicity.28–33 Herein, we report an electrochemical method to detect tumor exosomes. Different from most of the electrochemical assays, which achieve exosome detection via capturing exosomes on the surface of electrode,22-26 we use PSMA (prostate-specific membrane antigen) aptamer-magnetic bead bioconjugates to capture tumor exosomes derived from LNCaP cells and convert the exosomes detection to nucleic acid detection. Even though some reported methods of exosome detection have utilized aptamers. But the straightforward use of aptamers on the surface of electrode is difficult to get a signal amplification for exosome detection.26 In this project, three kinds of messenger DNAs (mDNAs) hybridized with the PSMA aptamers modified on magnetic bead, to capture tumor exosomes derived from LNCaP cells. In the presence of target exosomes, the aptamers recognize and bind to PSMA on the exosome membrane, and mDNAs are released. After magnetic separation, the released mDNAs are left in the supernatant. Then, an electrochemical method based on cyclic enzymatic amplification is used to detect the released mDNAs. Since one aptamer hybrizes with three mDNAs, one exosome/aptamer hybrid releases three mDNAs, and as a result triple the amount of mDNA can be released to amplify the signal. Furthermore, we combine the exonuclease III assisted target recycling strategy with multi-messenger DNAs (mDNAs), which could also greatly amplify the signal.34,35 The detection limit down to 70 particles/µL was achieved, which is lower than the LODs of most currently available methods.16,22,26,36,37 This assay can be used to detect tumor exosomes in complex biological samples, demonstrating potential application in real sample diagnosis.

EXPERIMENTAL SECTION Materials and Chemicals. Tris(2-carboxyethyl)

phosphine

hydrochloride

(TCEP,

98%)

and

tris-(hydroxymethyl) aminomethane were purchased from Aladdin (Shanghai, China).


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Bovine Serum Albumin (BSA) and 6-mercapto-1-hexanol (MCH) were obtained from Sigma-Aldrich

(St.

Louis,

N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide N-hydroxysuccinimide

(NHS)

and

MO,

USA).

hydrochloride

(EDC),

hexaammineruthenium(III)

chloride

were

purchased from Alfa Aesar (Massachusetts, USA). 1, 2-bis(dimethylamino)-ethane (TEMED), acrylamide mixed solution, ammonium persulfate (APS, 40%), low range DNA ladder, fetal bovine serum (FBS), RPMI-1640 medium and Dulbecco’s modified eagle medium (DMEM) were supplied by Sangon Biotechnology Co. Ltd. (Shanghai, China). Exonuclease III (Exo III) was purchased from New England Biolabs Co., Ltd. (Beijing, China). Carboxyl-modified magnetic beads were obtained from BaseLine Chromtech Research Centre (Tianjin, China). All other chemicals were of analytical reagent grade and used without further purification. All the HPLC-purified oligonucleotides used here were synthesized and purified by Sangon Biotech. Co., Ltd. (Shanghai, China), and all the oligonucleotide sequences are listed in Table S1. A Tris-HCl buffer (10 mM, pH 7.4) was used as the immobilization buffer. A phosphate buffer solution (PBS; 10 mM, pH 7.4) containing 0.1 M NaCl was employed as the washing buffer. A 0.1 M imidazole-HCl buffer (pH 7.4) was used to activate the carboxyl-modified magnetic bead. A 10 mM Tris-HCl buffer (pH 7.9) containing 10 mM MgCl2 and 250 mM KCl was employed as Exo III activity buffer. A Tris-HCl buffer (10 mM, pH 7.4) containing 5 µM Ru(NH3)63+ was used as the electrochemical buffer. Deionized water (18 MΩ/cm resistivity) obtained from a Millipore water system was used throughout the experiment. Instrumentation. Transmission electron microscopy (TEM) was performed on an H-7500 (HITACHI) transmission electron microscope operating at an accelerating voltage of 100 kV. The UV-vis

absorption

spectra

were

obtained

on

a

Cary

5000

UV-vis-NIR

spectrophotometer (Varian, USA). Polyacrylamide gel electrophoresis (PAGE) was performed on a JY600C electrophoresis apparatus (Beijing Junyi-dongfang electrophoresis equipment Co. Ltd., Beijing, China) and imaged on a Tanon-3500 gel image system (Shanghai, China). An ultracentrifuge OptimaTM XPN (Beckman Coulter, USA) was used to isolate exosomes. Quantization of exosomes was achieved using ZetaView (Particle Metrix, Germany). Differential pulse voltammetry (DPV) was carried out using a CHI760C electrochemical workstation (Chenhua, Shanghai, China).



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Cell Culture and Exosome Extraction. LNCaP cells and MCF-7 cells were grown in RPMI-1640 medium and HeLa cells were maintained in DMEM culture medium. Both culture media were supplemented with 10% FBS, 100 µg/ml streptomycin and 100 U/ml penicillin. All cells were cultured in a 37 oC incubator with 5% CO2 atmosphere. Exosomes were isolated from the conditioned media of serum-free cultures of different cancer cells as reported previously.

38,39

In brief, the conditioned medium from 106 cells was harvested after

48 h following serum-free defined medium renewal and centrifuged at 300 g for 10 min to eliminate cell contamination. The supernatant was further centrifuged at 16500 g for 30 min and filtered through a 0.22 µm filter. Then exosomes were pelleted by ultracentrifugation at 110 000 g for 2 h, washed with PBS, and pelleted again. Last, the sediment exosomes were resuspended in PBS and stored at -80 oC until use. Transmission Electron Microscopy (TEM). The TEM of the prepared exosomes was carried out via negative staining using a 2% solution of uranyl acetate (pH 4.5) according to previous protocol with modification.40 Briefly, 10 µL of exosomes in PBS was placed onto carbon-coated copper grids for 60 s; then the remaining solution was absorbed by filter paper. Next, 10 µL of uranyl acetate solution were placed onto the grids for another 60 s. After removing the stain, the prepared grids were dried at room temperature and imaged using an H-7500 transmission electron microscope. Preparation of Aptamer Modified Magnetic Beads. The

aptamer-magnetic

previously.

41,42

bead

bioconjugates

were

prepared

as

reported

Briefly, 100 µL of carboxyl-modified magnetic beads (particle size:

2~3 µm; loading capacity: ~500 µmol/g; concentration: 5 mg/mL) were first separated and washed three times with 0.5 mL imidazole-HCl buffer (pH 7.4) containing 0.02% Tween-20. Then magnetic beads were activated in 200 µL of 0.1 M imidazole-HCl buffer containing 10 mg of NHS and 20 mg of EDC for 1 h with gentle shaking at room temperature. After that, 5 µL 100 µM amino-modified aptamer was added into the activated MB solution, and the resulting mixture was allowed to react for 12 h at 37 oC with gentle shaking. In order to minimize nonspecific adsorption (for Figure S2B, it can be thought there is a nonspecific adsorption for the MBs about 20% compared with MBs in Figure S2A), after magnetic separation and three washes with 200 µL imidazole-HCl buffer, the aptamer-MBs were dispersed in 200 µL 0.1 M PBS (pH 7.4) containing 3% BSA, 0.1 M NaCl and 0.02% Tween-20 for 1 h at 37 oC,


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followed by three washes with PBS buffer (0.1 M, pH 7.4) and redispersed in 200 µL PBS buffer. The loading capacity of aptamer-MBs is about 1 µmol/g. Next, 150 µL of three mDNA (10-5 M for each) were added to the above solution for 2 h at 37 oC. After washing three times with PBS buffer, the hybridization complex was resuspended in 200 µL PBS buffer for further use. The final aptamer-mDNA-MBs solution was rather stable for 2 months when stored in a refrigerator at 4 oC. Immobilization of Probe DNA. Prior to immobilization, the 2 mm diameter gold electrode was pretreated according to a previous protocol.43 For the immobilization of P1 on the cleaned gold electrode surface, a mixture of 6 µL of TCEP solution (10 mM) with 6 µL P1 solution (10 mM) was first incubated for 1 h to reduce the disulfide bond and generate a free thiol group for the formation of an Au-S bond, followed by diluting the mixture to 100 µL with Tris-HCl buffer (10 mM, pH 7.4).44 Then, the cleaned electrode was incubated in the diluted P1 solution for 18 h at 4 oC. After that, the electrode was rinsed with Tris-HCl buffer and subsequently passivated with MCH (1 mM) for 2 h to block the unoccupied surface binding sites and displace nonspecifically bound P1 on the electrode surface.45 Next, the passivated electrode was rinsed three times with Tris-HCl buffer and scanned, and the obtained electrochemical signal was recorded as initial I (Iinitial). Finally, the electrode was thoroughly rinsed with Tris-HCl buffer and ready for DNA hybridization. Exosome Detection. A specified concentration of exosomes was added to 5 µL of a suspension of the aptamer-magnetic beads hybridization complexes, followed by diluting the mixture to 10 µL with PBS buffer. Then, the mixture was incubated at 37 oC for 2 h to release the mDNAs. After magnetic separation, the mDNAs were left in the supernatant and ready for detection. The mDNA detection was performed using a conventional three-electrode system, consisting of a saturated calomel (SCE) reference electrode, a platinum wire counter electrode, and a modified gold working electrode. Electrochemical signals were measured by DPV (-0.5 to +0.1 V; amplitude, 0.05 V; pulse width, 0.05 s; pulse period, 0.2 s) in electrochemical buffer. For mDNA detection, the supernatant containing mDNA mentioned above was dropped onto the inverted electrode with immobilized P1 and incubated at 37 oC for 2 h in a humidified container. After that, the electrode was rinsed with Tris-HCl buffer thoroughly. Then, the electrode was treated with Exo III at a concentration of 1 U/µL at 37 oC for 80 min.



Subsequently, the electrode was thoroughly rinsed with Tris-HCl buffer and scanned to obtain the final current (Ifinal). The electrochemical signal changes corresponding to the concentration of exosomes were calculated as ΔI = Iinitial－Ifinal. Error bars shown on individual figures correspond to variabilities among six independent trials of each experiment.

RESULTS AND DISCUSSION Characterization of Exosomes. The TEM image was used to examine the morphologies of the collected exosomes derived from LNCaP cells. As shown in Figure 1, these exosomes contain double-walled lipid membrane layers and their diameters are in the range of 40-100 nm, consistent with the size of reported exosomes.46 We also used nanoparticle tracking analysis (NTA) to confirm the size distribution of isolated exosomes (Figure S1). These results suggest that we did obtain exosomes and that the isolated exosomes could be used for further experiments.

Figure 1. Typical TEM images of the LNCaP cell- derived exosomes extracted from the conditioned medium. Mechanism of the Assay. Scheme 1 shows the mechanism of this strategy for tumor exosome detection. Two main processes are involved. One is the release of mDNAs triggered by the target exosomes (Scheme 1A) and the other is the Exo III-assisted target recycling signal amplification detection (Scheme 1B). As shown in Scheme 1A, the PSMA aptamer-mDNA complexes are immobilized on the surfaces of magnetic bead. PSMA,


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a biomarker enriched in LNCaP cells is also expressed in their exosomes.47,48 The introduction of LNCaP cell-derived exosomes changes the conformation of aptamer-mDNA complex as the aptamers bind to PSMA on the exosome membranes, and mDNAs are released. Because the amount of the mDNAs released is proportional to the number of target exosomes, we can determine the number of exosomes by quantifying the released mDNAs. In addition, since one aptamer hybrizes with three mDNAs, one exosome/aptamer hybrid releases three mDNAs, and as a result triple the amount of mDNA can be released to amplify the signal (Scheme 1A). It is worth noting that the sequence of mDNA consists of two parts: one complementary to part of the aptamer and another complementary to P1. After magnetic separation, the released mDNAs are left in the supernatant. As shown in Scheme 1 B, an electrochemical method based on cyclic enzymatic amplification is used to detect the released mDNAs. In this process, Exo III, a kind of sequence-independent enzyme, specifically catalyzes the stepwise removal of mononucleotides from the 3’-hydroxyl ends of DNA duplexes.35,49,50 When the released mDNAs bond to P1, double-stranded structures can be formed (Scheme 1B, b). Then, P1 of the double-stranded DNA is digested by Exo III, releasing the mDNAs, which then hybridize with another P1 to initiate a new cycle of Exo III cleavage reaction (Scheme 1B, c). Because of Exo III-assisted target recycling amplification, less P1 would be left on the surface of the gold electrode in a specified period. For detection, Ru(NH3)63+ is used as an electrochemical signal reporter, since it can bind to the negatively charged phosphate backbones of DNA and be electro-reduced to Ru(NH3)62+. Since Ru(NH3)63+ can quantitatively bind to DNA, the electrochemical signal is in proportion to the DNA on the electrode surface.51 Comparing scheme 1B a and d, it is obvious that the signal decreases, and the electrochemical signal change (ΔI=Ifinal-Iinitial) is proportional to the concentration of released mDNA, which is, in turn, proportional to the concentration of tumor exosomes.



Scheme 1. Schematic illustration of the detection of tumor exosomes: (A) Aptamers recognize and combine with exosomes and release mDNAs; (B) Detection the released mDNAs: (a) P1 modified gold electrode blocked with MCH, (b) Capture of mDNAs, (c) Treated with Exo III exonuclease, (d) DPV measurement in the electrochemical buffer. Feasibility of This Strategy. In order to verify the feasibility of this strategy, we recorded the native polyacrylamide gel electrophoresis (PAGE) and the electrochemical signals under different conditions. PAGE was used to characterize the formation of aptamer-mDNA complexes and Exo III function. As shown in Figure 2A, lane 6, The aptamer-mDNAs migrated more slowly than aptamers, M1, M2 and M3 (Figure 2A, lanes 2, 3, 4, 5), indicating the formation of aptamer-mDNA complexes. But the hybridization efficiency did not reach 100%. In order to improve the hybridization efficiency, excess mDNAs (3 equiv) have been added in the process of hybridization. In addition, Figure 2A, DPV measurements were recorded with the prepared electrode at different stages. As shown in Figure 2B, when P1 was immobilized on the gold electrode, followed by MCH blocking (step a, Scheme 1B), there is a DPV signal for the reduction of Ru(NH3)63+(Figure 2B, a). After incubating the extended gold electrode with mDNAs (released from aptamer-mDNA complex incubated with 20 000 LNCaP cell-derived exosomes, Scheme 1A), the DPV signal increased (Figure 2B, b). After


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treating the working electrode with Exo III, the signal decreased sharply because P1 strands were digested by Exo III cyclically, leaving less Ru(NH3)63+ electrostatically bound to the negatively charged phosphate backbones of DNAs (Figure 2B, c). These results indicate the viability of the proposed strategy for determination of tumor exosomes. We also determined whether aptamers were bound to magnetic bead using UV-vis absorption spectroscopy (see Figure S2). As illustrated in Figure S2, the UV-vis absorbance of aptamer in the supernatant was obviously decreased after the aptamers reacted with MBs for 2 h, indicating that the aptamers had bonded to magnetic bead successfully. All the results suggested that the aptamer recognition-induced multi-DNA release and the cyclic enzymatic amplification for exosome detection is reliable.

Figure 2. (A) Native PAGE gel of different samples. Lane1: the standard DNA markers of 10, 15, 20, 25, 35, 50, 75, 100,150, 200, and 300 bp of DNA sequences; Lane 2: 5 µM aptamers; Lane 3: 5 µM M1; Lane 4: 5 µM M2; Lane 5: 5 µM M3; Lane 6: 5 µM aptamers hybridized with M1, M2 and M3 at 37 oC for 2 h; Lane 7: 5 µM P1; Lane 8: 5 µM P1 hybridized with M1 at 37 oC for 2 h; Lane 9: lane 8 after treatment with 1 U/µL Exo III. (B) DPV signals of the gold electrode at different stages (a) MCH-blocked P1-modified gold electrode; (b) after capture of mDNAs; (c) after digestion by Exo III (1 U/µL). (DPV parameters: −0.5 V to +0.1 V; amplitude, 0.05 V; pulse width, 0.05 s; pulse period, 0.2 s). Optimization of Experimental Conditions. In order to achieve the best performance for detecting tumor exosomes, the experimental conditions, including the concentration of P1 and the cleavage time of Exo III were investigated. For the P1 concentration optimized, P1 with different



concentrations were immobilized on the gold electrode. The concentration of M1 was fixed at 10 µM. Following hybridization between the P1-modified gold electrode and M1, the Ru(NH3)63+ reduction current was recorded. As depicted in Figure 3A, the current increased with increasing concentrations of P1 from 0.2 to 0.6 µM and reached a peak value at 0.6 µM. Further increase in the concentration decreased the hybridization efficiency, indicating that the optimal P1 concentration was 0.6 µM. Since the target molecule is recycled, the reaction time of Exo III is a key factor in our strategy. In this work, 10 µL 2 µM M1 was dropped on the P1-modified electrode (0.6 µM) and incubated at 37 oC for 2 h. After that, 1 U/µL Exo III was added to the reaction system and incubated ay 37 oC for a specified period of time ranging from 20 to 100 min. Subsequently, the electrochemical signal was recorded and plotted against the time of incubation. As shown in Figure 3B, the current decreased with increasing time from 0 to 80 min, reaching a minimum value after 80 min, Therefore, the optimal time for reaction with Exo III was 80 min.

Figure 3. (A) Optimization of P1 probe concentration used for self-assembly on the gold electrode. (B) The dependence of the peak current of the P1/M1 hybrid-modified electrode on the time for digestion by Exo III (1 U/µL). LNCaP Cell-derived Exosome Detection. Under the optimized conditions, LNCaP cell-derived exosomes with different concentrations were added to the reaction system to characterize the detection range and sensitivity of this assay. As shown in Figure 4A, the DPV current decreased with increasing exosome concentration, indicating that with increasing exosome concentration, more mDNAs were released, thus more target molecule can be recycled, resulting in a decrease in DPV current (step e, Scheme 1B). There is a linear relationship between the variation of the electrochemical signal and the logarithm of


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the number of target exosomes in the range of 1000- 120000 with a correlation coefficient (R2) of 0.994 (Figure 4B). The linear fitting equation is ΔI = -0.1071 + 0.0462 lg[n], where ΔI is the signal change, defined as ΔI = Iinitial－Ifinal, where Iinitial and Ifinal represent the DPV currents of the P1-modified electrode before and after the electrode was incubated with released mDNA and Exo III, and n is the number of target exosomes. The sensitivity of this assay was calculated as 100.0462 µA/particle and the limit of detection was experimentally estimated to be 70 particles/µL, which is lower than the LODs of most of the current available methods (listed in Table 1). In addition, our detection platform is much simpler and uses less costly instruments than the other methods.

Figure 4. (A) Effect of exosome number on the DPV response. The number of exosomes is (a) 1000, (b) 2000, (c) 4000, (d) 8000, (e) 20000, (f) 40000, (g) 60000, and (h) 120000. (B) Linear relationship between the DPV peak current change and the logarithm of the number of exosomes secreted from LNCaP cells. (DPV parameters: −0.5 V to +0.1 V; amplitude, 0.05 V; pulse width, 0.05 s; pulse period, 0.2 s). Table 1. Comparison of LODs of currently available methods for exosome determination. No.

Method

LOD (particles/µL)

Reference

1

Fluorescence method

50

21

2

Nano-plasmonic sensor

3×103

19 5

3

UV−vis absorbance method

5.2×10

4

UV−vis absorbance method

2760

5

Electrochemical Immunosensor

2×102

22

3

26

6

Electrochemical method

1×10

7

Electrochemical method

70

34 35


This work


In order to investigated the stability of the P1-modified electrode, we detected the signal changes of P1 DNA on the electrode before and after incubating the MCH-blocked P1-modified gold electrode with PBS and subsequent Exo III, and calculated the relative error of the signal change, using eqn:

Relative Error =

|‫ܫ‬′ሺfinalሻ − ‫ܫ‬′ሺinitialሻ| × 100% ‫ܫ‬′ሺinitialሻ

where Iˊ(initial) and Iˊ(final) represent the DPV currents of the MCH-blocked P1-modified electrode before and after incubating with PBS and subsequent Exo III. we tested the relative error with six independent determinations. The average relative error was 1.72%, indicating the good stability of P1-modified electrode. Moreover, the reproducibility of the biosensor was tested by detecting 20000 particles with six electrodes prepared independently with the relative standard deviation (RSD) of 3.97 %, indicating an acceptable reproducibility of our strategy. Therefore, the present method can be used for efficient and convenient quantitative analysis of LNCaP cell-derived exosome.

Selectivity of the proposed assay. A pivotal issue is the specificity of the proposed strategy. Consequently, experiments were conducted on MCF-7 cell-derived exosomes and HeLa cell-derived exosomes with the same number of particles (40 000) as LNCaP cell-derived exosomes. As displayed in Figure 5, the signal changes obtained from MCF-7 cell-derived exosomes and HeLa cell-derived exosomes were much smaller than the current change for LNCaP cell-derived exosome detection. The result is in accordance with the fact that only LNCaP cell-derived exosomes express PSMA antigen and also proved that the proposed assay has good selectivity due to the high affinity of the aptamer.


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Figure 5. DPV signal changes in response to exosomes secreted from LNCaP cells, MCF-7 cells and HeLa cells. Determination of Exosomes in Complex Biosamples. To evaluate the clinical utility of this proposed method in detecting tumor exosomes, we quantified the LNCaP cell-derived exosomes in fetal bovine serum (FBS). Serum contains a variety of highly concentrated components in addition to exosomes, so we introduced ultra-centrifuged (UC) FBS as a mimetic clinical system to evaluate the performance of this detection platform. As shown in Figure 6, the result obtained from exosomes in PBS was almost the same as that in UC FBS (30% and 60%). Therefore, the results indicated that the proposed assay can work well in complex biosamples and holds great potential for future use in clinical samples.

Figure 6. Detection of LNCaP cell-derived exosomes in different environments. UC FBS represents the ultracentrifuged fetal bovine serum.



CONCLUSION In conclusion, a sensitive and specific method was designed to detect LNCaP cell-derived exosomes. The present detection method has a dual signal amplification: release of multiple DNAs and Exo III-assisted target recycling amplification. Moreover, our method has tremendous advantages, such as the utilization of aptamers instead of antibodies and electrochemical detection, thus decreasing the cost. This assay can determine as low as 70 particles/µL with a linear (log scale) of 1000-120000 particles/µL. This proposed strategy shows great potential for exosome detection, which sheds a new light on the efficient cancer diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Sequences of the oligonucleotides used in this study (Table S1) and Measurement of LNCaP cell-derived exosomes by Nanoparticle Tracking Analysis (NTA) (Figure S1) and UV-vis absorption of aptamer and aptamer1 in the supernatant before and after aptamers connected with MBs (Figure S2).

AUTHOR INFORMATION *Corresponding authors: *†E-mail: [email protected]. *‡E-mail: [email protected]

ACKNOWLEDGMENTS This work is supported by NSFC (21375063, 21575069, 21675088 and 21335004), the Program for Outstanding Innovation Research Team of Universities in Jiangsu Province and Priority Academic Program Development of Jiangsu Higher Education Institutions.

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