Article pubs.acs.org/ac
Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Dichroic Mirror-Assisted Electrochemiluminescent Assay for Simultaneously Detecting Wild-type and Mutant p53 with Photomultiplier Tubes Yupeng He, Fang Zhang, Bin Zhang, and Guizheng Zou* School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, China S Supporting Information *
ABSTRACT: The electrochemical-dependent and unstable intensity of electrochemiluminescence (ECL) makes it difficult to identify ECL in a waveband-resolved way on photomultiplier tube (PMT)-based traditional ECL analyzers. Herein, a dual-color ECL strategy is proposed by transferring ECL to two PMTs in a waveband-resolved way via dichroic mirror, simultaneously detecting wild-type p53 (WTp53) in near-infrared wavebands with CdTe (λmax = 782 nm) nanocrystals as tag and mutant p53 (MUp53) in eye-visible wavebands with CdSe (λmax = 554 nm) nanocrystals as tag. The two targets can be color-selectively determined from 10 fM to 100 pM with a limit of detection at 5 fM for MUp53 and from 50 fM to 100 pM with a limit of detection at 10 fM for WTp53, respectively. The dichroic mirror-involved ECL setup is easy to assemble for popularization, which would not only eventually enable PMT-based multiple-color ECL analysis but also make it is possible to directly determine the changed level of tumor suppressors for cancer diagnosis and therapeutic evaluation via one-pot ECL assay.
E
and Bard;11 they successfully determined the ECL spectrum of many NCs with a charge-coupled device (CCD) camera and grating spectrometer.12−18 Our group has been involved in acquiring technology of the ECL spectrum for more than ten years.19,20 A homemade ECL spectral analyzer with electrochemical analyzer as upper monitor and CCD cameracombined grating spectrometer as spectral detector was proposed for accurately recording the ECL spectrum,21−23 and a spectrum-based ECL sensing strategy with NCs as tags was also achieved in our group.24−26 Importantly, corresponding research demonstrated that both dual-stabilizer-capped CdTe and CdSe NCs can preserve their high-passivated surface states and are promising monochromatic ECL tags for spectralresolved assays.24,25 Consequently, a spectrum-based dual-color ECL immunoassay for simultaneously and selectively determining two targets was achieved on the homemade ECL spectral analyzer with the dual-stabilizer-capped CdTe and CdSe NCs as tags.27 Unfortunately, compared with PMT-based traditional ECL setups, the CCD camera-combined grating spectrometer is complicated and too expensive to popularize the spectral ECL assay,28,29 let alone the specially designed light path, synchronously triggering monitoring for the waveband-resolved ECL assay.21,23,26 Novel simple and easy-to-assemble ECL setups with low cost are urgently required for the development of waveband-resolved ECL assays.
lectrochemiluminescence (ECL) is the controllable chemiluminescence (CL) generated via electrochemical redox.1−3 ECL analysis is superior to fluorescence analysis in terms of signal-to-noise ratio and its simple setup,1 and ECLrelated bioassays have excellent reproducibility, sensitivity, and a desirable limit of detection (LOD).2,3 A series of commercial ECL analyzers and Ru(bpy)32+/tri-n-propylamine (TPrA) reagent kits have been developed by Roche Diagnostics and used for biomedical and diagnostics assays.1 The initial investigation on ECL setups was achieved by Bard4 in which ECL of 9,10-diphenylanthracene under varied electrochemical conditions was successfully recorded with a Dumont 6467 photomultiplier tube (PMT) as detector. From then on, PMT was extensively utilized in traditional ECL setups due to its high sensitivity, small dark current, large signal-to-noise ratio, and fast response nature.1 PMT-based traditional ECL setups are easy to assemble and operate; the overwhelming majority of ECL research and commercial-available ECL tests are conducted on them.1−3,5−7 Unfortunately, ECL is related to electrochemical redox and unstable in a time-resolved way,5 which makes it difficult to define the ECL spectrum in a wavelength-scanning manner similar to that of fluorescence.8 From this point, the traditional ECL setups are designed by directly placing a working electrode in front of PMT or other photodetectors without a dispersion device1,9,10 and fails to meet the demands for waveband-resolved multiplexing ECL assays and convenient ECL spectrum acquisition. To the best of our knowledge, the promising breakthrough toward waveband-resolved ECL assays was achieved by Ding © XXXX American Chemical Society
Received: February 20, 2018 Accepted: March 21, 2018
A
DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
of the present test system have been detailed in our previous papers.27 The waveband-resolved ECL assay was conducted on a homemade ECL detecting system with CHI1040C multichannel electrochemical analyzer (Shanghai Chenhua Co., Ltd., China) as upper monitor (Scheme 1) and two H10721−20 PMTs (Hamamatsu Photonics K.K., Japan, 230−920 nm) equipped with C10709 power supplies (Hamamatsu Photonics K.K., Japan, controllable output voltage, −5 to 5 V) as detectors. The ECL generated at the working electrode was collected with an objective lens, turned to directional light via the collimation lens, and finally transferred to two PMTs via dichroic mirror in a waveband-resolved way. The photocurrents recorded on the two PMTs were transferred to two auxiliary channels of the upper monitor. The electrochemical signal (via the main channel 1) and photocurrent signal from two PMTs (via the auxiliary channels 2 and 3) was synchronously detected by CHI1040C multichannel electrochemical analyzer. DMLP638R dichroic mirror (Thorlabs, Inc., cutoff wavelength 638 nm) was adopted to reflect ECL with a wavelength shorter than 638 nm (i.e., ECL from CdSe554) to one PMT (channel 3) and transmit ECL with a wavelength longer than 638 nm (i.e., ECL from CdTe782) to the other PMT (channel 2). A three-electrode system was involved in this case, including an Au working electrode (diameter of 5 mm), a Pt coil counter electrode, and a Ag/AgCl (saturated KCl) reference electrode. ECL spectra acquisition was accomplished on a homemade ECL spectrometer consisting of an Acton SP2300i monochromator equipped with a liquid N2-cooled PyLoN 400BReXcelon digital CCD detector (Princeton Instruments, U.S.A) and a VersaSTAT 3 electrochemical analyzer (Princeton Applied Research, U.S.A.).24,25 The TTL strategy was adopted to simultaneously trigger VersaSTAT 3 electrochemical analyzer and CCD camera-combined Acton SP2300i monochromator; ECL generated at working electrode surface was collected with an objective lens and then delivered to the Acton SP2300i monochromator. Procedures for Dual-Color ECL Analysis. According to Scheme 2, the dual-color ECL analysis was in a sandwich-type
It is well-known that dichroic mirrors have been extensively utilized in waveband-resolved optical devices for fluorescence bioassays30−33 because dichroic mirrors can physically transmit light of a given waveband and reflect light of another waveband for dual-view imaging and dual-color assays.34 Herein, a promising waveband-resolved ECL setup is designed by transferring ECL to two PMTs with a dichroic mirror in a waveband-resolved way (Scheme 1), synchronously recording Scheme 1. Scheme of Dichroic Mirror-Assisted ECL Setup
the photocurrent of two PMTs (via channels 2 and 3, respectively) and the electrochemical current (via channel 1) with a multichannel electrochemical analyzer as upper monitor (Scheme 1 and Experimental Section) was achieved in an easyto-assemble way. Importantly, the homemade ECL setup is easy to operate, and a dual-color ECL assay is achieved on it for simultaneously determining wild-type p53 (WTp53) gene35−37 in near-infrared band with dual-stabilizer-capped CdTe (λmax = 782 nm, named CdTe782) NCs as tag and mutant p53 (MUp53) gene38−41 in eye-visible waveband with dualstabilizer-capped CdSe (λmax = 554 nm, named CdSe554) NCs as tag. It is well-known that WTp53 is an important tumor suppressor related to the cell cycle and programmed cell apoptosis; this work demonstrates the promising possibility of PMT-based multiplexing ECL assays for cancer diagnosis and therapeutic evaluation in a waveband-resolved way similar to the fluorescence multiplexing assay with several NCs as tags.42
Scheme 2. (A) Labeling and (B) Fabricating Procedure for Dual-Color ECL Assay
■
EXPERIMENTAL SECTION Chemicals and Materials. All chemical reagents were of analytical grade or better and used as received (see Supporting Information). CdTe782 and CdSe554 NCs were prepared by the previously reported dual-stabilizer-capped strategy with desired refluxing time.27,29,43 DNA sequences were obtained from Sangon Biological Engineering Technology & Services Company Ltd. (Shanghai, China) and purified with highperformance liquid chromatography. Capture DNA probe for WTp53 (S1WTp53): 5′-triple SH-GTAACTGCATTTCTTCACCTG-3′. Target DNA WTp53 (S2WTp53): 5′-CGTGGAGCTACAGTGGCTGTAAAGCAGGTGAAGAAATGCAGT-3′. Signal DNA for WTp53 (S3WTp53): 3′-SH-(CH2)6-CCAGCACCTCGATGTCAC-5′. Capture DNA probe for MUp53 (S1MUp53): 5′-triple SH-AAACCTCAACTTTCCCATTTT-3′. Target DNA MUp53 (S2MUp53): 5′-CAGCAGAGGTCAAAGAGGAGGTGAAAAATGGGAAAGTTGAGG-3′. Signal DNA for MUp53 (S3MUp53): 3′-SH-(CH2)6-ATTGTCGTCTCCAGTTTC-5′. Apparatus. The instruments and methods used for common optical, electrochemical, and ECL characterization
procedure by immobilizing probe DNA onto the gold surface via tridentate anchoring for desired structural stability of the fabricated biosensor.44,45 CdSe554-labeled signal DNA for MUp53 (i.e., S3MUp53| CdSe) and CdTe782-labeled signal DNA for WTp53 (i.e., S3WTp53|CdTe) were prepared by forming Cd−S bonds between the terminal thiols of signal DNA and cadmium ions B
DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry on the NC surface (Scheme 2A).29,38,43,46 For example, a drop of 50 μL signal DNA for MUp53 (1 × 10−5 M) was mixed with 2 μL of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to cut the S−S bond and then incubated with 450 μL of colloidal CdSe NCs (1.6 mg/mL) for 24 h at 4 °C to form the Cd−S bond. The obtained S3MUp53|CdSe was finally incubated with 1 wt % bovine serum albumin (BSA) at 4 °C for 1 h to block the nonspecific active and binding sites. The Au electrode was polished with 0.5 μm alumina, electrochemically cleaned in 0.5 M H2SO4 by scanning potential from −0.3 and 1.6 V, washed with water, and finally dried with nitrogen. The freshly prepared Au electrode was incubated in a drop of 200 μL pH 7.0 TE buffer (10 mM Tris, 1.0 mM EDTA, 1.0 M NaCl, 1.0 mM TCEP) containing 0.5 μM S1MUp53 and 0.5 μM S1WTp53 for 4 h at 37 °C. The obtained ⎧ S1
Au-⎨S1MUp53 was immersed in 1 mM 3-mercapto-1-hexanol ⎩
WTp53
Figure 1. (A) Absorption (a, c) and PL spectra (b, d) of monodispersed CdSe554 (green line) and CdTe782 (red line) in aqueous medium. (B) ECL spectra of monodispersed CdSe554 (a, green line), CdTe782 (b, red line), and the surface-confined NCs of
(MCH) for 1 h for blocking and then incubated with a drop of 200 μL TE buffer containing both MUp53 and WTp53 of designed concentrations for 2 h at 37 °C. The resultant Au-
⎧ S1MUp53 − MUp53 ⎨ S1 ⎩ WTp53 − WTp53
⎧ S1
was incubated in 200 μL solution containing
S3MUp53|CdSe and S3WTp53|CdTe for 2 h at 37 °C to form Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
(Scheme 2B). Control experi-
− MUp53 − S3
WTp53
|CdSe
WTp53
V at 50 mV/s in 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM (NH4)2S2O8 and 100 mM KCl.
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ . ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
■
RESULTS AND DISCUSSION Optical and ECL Spectra of CdSe554 and CdTe782 in Aqueous Medium. According to Figure 1, the as-prepared CdSe554 displayed an absorption peak at ∼526 nm (Figure 1A, curve a) and a symmetrical narrow PL peak at ∼554 nm (Figure 1A, curve b). CdSe782 NCs displayed an absorption peak at ∼738 nm (Figure 1A, curve c) and a nearly symmetrical narrow PL peak at ∼782 nm (Figure 1A, curve d). The ECL spectrum of the monodispersed CdSe554 displayed a narrow sole peak around 554 nm, whereas the ECL spectrum of the monodispersed CdTe782 NCs display a symmetric sole peak around 782 nm (curves a and b, Figure 1B). The ECL spectra of both CdSe (554) and CdTe (782) are almost identical to corresponding PL spectra, respectively, indicating these NCs are highly passivated21,24,25,28 and are promising for dual-color ECL assay via complicated labeling and biorelated reactions.27 Actually, the surface-confined CdSe554 and CdTe782 in Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
− MUp53 − S3
|CdSe
whole ECL process. Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe was WTp53 ⎩ WTp53 formed with sample containing both 0.5 pM MUp53 and 0.5 pM WTp53.
scanning Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe from 0.0 to −1.60 ⎩
|CdSe
⎧ S1
ments were prepared in a similar fashion. The proposed dual-color ECL assay was carried out by ⎧ S1
− MUp53 − S3
Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe (c, black line) in 50 mM TrisWTp53 ⎩ WTp53 HCl buffer (pH 8.0) containing 100 mM (NH4)2S2O8 and 100 mM KCl by scanning potential from 0 to −1.60 V for one cycle at 50 mV/s. The ECL spectra resulted from the total photons generated during the
⎧ S1
− MUp53 − S3
The fabricating procedure of
|CdSe
Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe was characterized stepwise ⎩
WTp53
WTp53
with the Fe(CN)63−/Fe(CN)64− redox couple (Figure S1). A well-defined cyclic voltammetry (CV) curve with a peak potential separation of 69 mV was obtained on bare Au (Figure S1, curve a). The successive immobilization of S1MUp53 and S1WTp53 (curve b in Figure S2) in step 1 of Scheme 2B, MUp53 and WTp53 (curve c in Figure S2) in step 2 of Scheme 2B, and S3MUp53|CdSe and S3WTp53|CdTe (curve d in Figure S2) in step 3 of Scheme 2B onto the Au surface brought out gradually enlarged peak potential separation and decreased current intensity because forming biomolecules complexed at the electrode surface would block the charge transfer.24,25,27 Waveband-Resolved ECL and Electrochemical Response of Surface-Confined CdSe554 and CdTe782. ⎧ S1
− MUp53 − S3
|CdSe
Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe can bring out ECL with ⎩
WTp53
WTp53
efficient photoelectric response on two PMTs for separately and simultaneously recording the reflected ECL of CdSe554 with a wavelength shorter than 638 nm and the transmitted ECL of CdTe782 with a wavelength longer than 638 nm (Figure 2A and B). These results not only confirmed that CdSe554 and CdTe782 were simultaneously immobilized onto the Au surface via the proposed strategy (Scheme 2) but also provided unambiguous evidence that the homemade ECL setup can synchronously record electrochemical signal via the main signal channel 1 (Figure 2C) and photocurrent signal generated on two PMTs via the auxiliary channels 2 and 3 (Figure 2). The strong reductive current around −0.95 V in Figure 2C was mainly derived from the reduction of coreactant S2O82−
can preserve their monochro-
matic spectral ECL nature with efficient maximum emission around 554 and 782 nm (curve c, Figure 1B), respectively. The DMLP638R dichroic mirror is consequently chosen for the waveband-resolved ECL analysis as DMLP638R can reflect ECL of CdSe554 to one PMT (via channel 3) without interference from ECL of CdTe782 and simultaneously transmit ECL of CdTe782 to the other PMT (via channel 2) without interference from ECL of CdSe554 (Figure 1B). According to Scheme 2, both CdSe554 and CdTe782 were simultaneously immobilized on the Au surface by forming two sandwich-type DNA complexes, i.e. AuC
DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 3. (A) Electrochemical and (B) ECL response of surfaceconfined CdSe554 (a, black line recorded by channel 3) and CdTe782 (b, red line recorded by channel 2) of Au-
Figure 2. ECL of surface-confined (A) CdSe554 in Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ recorded by channel 3, (B) ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe ⎧ S1 − MUp53 − S3 |CdSe CdTe782 in Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe recorded by WTp53 ⎩ WTp53
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ in N2-saturated 50 mM Tris-HCl ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
buffer (pH 8.0) containing 100 mM (NH4)2S2O8 and 100 mM KCl by continuously scanning the potential from 0.0 to −1.6 V for five cycles
channel 2, and (C) corresponding electrochemical response of Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ recorded by channel 1 in N2⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
⎧ S1
saturated 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM (NH4)2S2O8 and 100 mM KCl at 50 mV/s. Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ was formed with a sample ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
because the direct electrochemical response of dual-stabilizercapped CdTe and CdSe NCs have been proven to be very weak.21,24,25,29,47 Although the electrochemical response of Audecreased dramatically in the
successive scan due to the consumed coreactant at the Au surface (Figure 3A), stable ECL for surface-confined both CdSe554 and CdTe782 in
⎧ S1 − MUp53 − S3 |CdSe Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe WTp53 ⎩ WTp53
with desired structural stabil-
(1)
CdSe NCs + ne → nR (CdSe)−•
(2)
nR (CdTe)−•
(3)
SO4 −• + R (CdSe)−• → R (CdTe)* + SO4 2 −
(4)
SO4 −• + R (CdTe)−• → R (CdTe)* + SO4 2 −
(5)
R (CdSe)* → CdSe + hv ( ∼554nm)
(6)
R (CdTe)* → CdTe + hv ( ∼782nm)
(7)
Research on spectrum-based dual-color ECL immunoassay with dual-stabilizer-capped CdTe and CdSe NCs as tags has proven that the possible energy transfer between the highly excited state CdSe* and the ground state CdTe NCs is negligible for surface-confined CdTe and CdSe NCs.27 The Au-
ity.44,45 Finally, the dual-stabilizer-capped CdTe and CdSe NCs, i.e., CdTe782 and CdSe554, are electrochemically stable, and the excited states of them can be relaxed to ground states via ECL21,25,27 so that both CdTe782 and CdSe554 could be repetitively reduced for stable reductive-oxidation ECL in a coreactant way. A typical reductive-oxidation mechanism is believed for the generation of ECL from Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ /S2O42−.27 ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
S2 O82 − + e → S2 O83 −• → SO4 2 − + SO4 −•
CdTe NCs + ne →
was achieved with prolonged scan for five cycles (Figure 3B). The reason might lie in three aspects. First, electron-injecting related reduction processes of dual-stabilizer-capped CdTe and CdSe NCs are the rate-determining steps for ECL; the limited coreactant molecules in the diffusion layer are enough for the generation of ECL.24,25 Second, immobilizing probe DNA onto the gold surface via tridentate anchoring can enable the Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
|CdSe
and negatively charged NCs radicals (R−•), i.e., both R(CdSe)−• and R(CdTe)−•, are produced via electrochemically reducing coreactants and NCs, respectively (eqs 1−3). Then, SO4−• radicals inject holes into the highest occupied molecular orbital (HOMO) of R−•, producing an excited state R*, i.e., both R(CdSe)* and R(CdTe)* (eqs 4 and 5). Finally, dual-color ECL is generated along with the relaxation processes from the excited species R(CdSe)* and R(CdTe)*, respectively (eqs 6 and 7).
containing 0.5 pM MUp53 and 0.5 pM WTp53.
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
− MUp53 − S3
at 50 mV/s. The Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe was formed WTp53 ⎩ WTp53 with sample containing both 0.5 pM MUp53 and 0.5 pM WTp53.
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ /S2O42− ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
ECL system was then
adopted for waveband-resolved ECL analysis with PMTs as detectors. Color Selectivity of the PMT-Based Dual-Color ECL Sensing Strategy. The color selectivity of the proposed waveband-resolved ECL analysis is first characterized by Au-
Upon the cathodic
potential scanning, strong oxidant SO4−• (E0 > 3.15 V vs SCE)1 D
DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry ⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
containing
MUp53
based and dual-waveband-resolved ECL strategy was further evaluated by simultaneously determining MUp53 and WTp53
formed with sample merely
or
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 ⎧ S1MUp53 ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
WTp53,
i.e.,
or
⎧ S1
Au-
− MUp53 − S3
|CdSe
with Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe , which were formed WTp53 ⎩ WTp53 with samples containing both MUp53 and WTp53 of the same
Au-
⎧ S1
− MUp53 − S3
|CdSe
concentration. The Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe disWTp53 ⎩ WTp53 played increased ECL via both channels 3 and 2 along with increasing concentration of MUp53 and WTp53 (Figure S2). Importantly, the maximum ECL intensity of Au-
(Figure 4). As for Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
recorded by channel 3 in-
creased linearly with logarithmic increasing concentration of MUp53 from 10 fM to 100 pM with an LOD of 5 fM, whereas ⎧ S1
− MUp53 − S3
|CdSe
the ECL of Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe recorded by WTp53 ⎩ WTp53 channel 2 increased linearly with logarithmic increasing concentration of WTp53 from 50 fM to 100 pM with an LOD of 10 fM. Notably, according to Figure 5B, no significant
Figure 4. Concentration-dependent maximum ECL intensity of Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ formed with samples merely ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
containing (A) MUp53 and (B) WTp53. The ECL was conducted by scanning potential from 0 to −1.60 V vs Ag/AgCl for one cycle in N2-saturated 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM (NH4)2S2O8 and 100 mM KCl at 50 mV/s, and the maximum ECL intensity was measured by (a, red line) channel 2 and (b, black line) channel 3, respectively. ⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ , ⎩ S1WTp53
obvious and increased ECL
was recorded by channel 3 along with increasing concentration of MUp53 (Figure 4A, curve b); only negligible ECL was recorded by channel 2 (Figure 4A, curve a) because ECL from CdSe554 was efficiently reflected to the PMT connected to channel 3 by DMLP638R. As for Au-
⎧ S1MUp53 ⎨ , ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
Figure 5. Concentration-dependent maximum ECL intensity of Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ formed with samples containing ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
MUp53 and WTp53 of the same concentration. The ECL was conducted by scanning potential from 0 to −1.60 V vs Ag/AgCl for one cycle in N2-saturated 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM (NH4)2S2O8 and 100 mM KCl at 50 mV/s, and the maximum ECL intensity was measured by (A) channel 3 for determining MUp53 and (B) channel 2 for determining WTp53.
obvious and increased ECL
was recorded by channel 2 along with increasing concentration of WTp53 (Figure 4B, curve a); only negligible ECL was recorded by channel 3 (Figure 4B, curve b) because ECL from CdTe782 was efficiently transmitted to the PMT connected to channel 2 via DMLP638R. From this point, it is clear the DMLP638R-assisted ECL system is qualified for wavebandresolved ECL assays with desired color selectivity. A linear relation between the maximum ECL intensity and logarithmic concentration of MUp53 was obtained from 10 fM to 100 pM via channel 3 (yellow dot-marked section for curve b in Figure 4A) with an LOD of 5 fM for determining MUp53. Another linear relation between the maximum ECL intensity and logarithmic concentration of WTp53 was obtained from 50 fM to 100 pM via channel 2 (yellow dot-marked section for curve a in Figure 4B) with an LOD of 10 fM for determining ⎧ S1
− MUp53 − S3
difference was obtained between the maximum ECL intensity (detected via channel 2) for WTp53 at 0 and 5 fM levels. The ⎧ S1
− MUp53 − S3
|CdSe
sensing performance of Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe for WTp53 ⎩ WTp53 simultaneously determining MUp53 and WTp53 was identical to the single analyte sensing performance of Au-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53
for determining MUp53 and
⎧ S1
Au-⎨S1MUp53 − WTp53 − S3 ⎩
WTp53
WTp53|CdTe
for determining WTp53,
respectively. Importantly, according to Figures 4 and 5, the ⎧ S1
− MUp53 − S3
|CdSe
ECL intensity of Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe recorded WTp53 ⎩ WTp53 by channel 3 was almost identical to that of Au-
|CdSe
WTp53. It is clear that Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe WTp53 ⎩ WTp53 would be capable of simultaneously determining MUp53 and WTp53 on the homemade ECL setup. Waveband-Resolved ECL Assay for Simultaneously Determining WTp53 and MUp53 with PMTs. The multiplexing sensing performances of the proposed PMT-
⎧ S1MUp53 − MUp53 − S3MUp53|CdSe ⎨ ⎩ S1WTp53
recorded by channel 3 with
MUp53 of the same concentration, whereas the ECL intensity ⎧ S1
− MUp53 − S3
|CdSe
of Au-⎨S1MUp53 − WTp53 − S3 MUp53|CdTe recorded by channel 2 WTp53 ⎩ WTp53 E
DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry was
almost
identical
⎧ S1MUp53 ⎨ ⎩ S1WTp53 − WTp53 − S3 WTp53|CdTe
to
that
of
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Au-
recorded by channel 2 with
WTp53 of the same concentration. Regardless, it is clear that the waveband-resolved ECL strategy is capable of multiplexing ECL assay for simultaneously determining two targets with desired sensitivity and color selectivity. As serials of combined dichroic mirrors can spectrally separate light emission into several different light beams with desired waveband width and then transfer them into different PMTs, this work might provide a promising candidate for waveband-resolved ECL multiplexing assays and easy-tooperate ECL spectrum acquisition with PMTs as detectors.
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CONCLUSIONS A simple and easy-to-popularize ECL setup was designed by dividing ECL into two light-beams of different wavebands with a dichroic mirror and then transferring them to different PMTs in a color-resolved way. A waveband-resolved ECL assay for simultaneously determining different targets with PMTs as detectors was achieved for the first time, which could be extended to simultaneously identifying multiple analytes, such as RNA, protein, and virus, with advantages of timesaving, minimized sample volume, and decreased cost. The dichroic mirror-assisted ECL setup would not only eventually enable PMT-based multiple-color ECL analysis but would also make it is possible to directly determine the changed level of tumor suppressors for cancer diagnosis and therapeutic evaluation via ECL technology. Importantly, as dichroic mirrors have been extensively utilized in dual-view optical devices for fluorescence assays and imaging, this work also provides a path toward dualview ECL assays and/or imaging besides the wavebandresolved assay via ECL intensity.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00831. Materials, electrochemical characterization for fabricating procedures, and ECL profiles for waveband-resolved dual-color assays (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; Tel: +86-531-88361326; Fax: +86-531-88564464. ORCID
Yupeng He: 0000-0002-4582-3129 Bin Zhang: 0000-0002-1529-6356 Guizheng Zou: 0000-0002-3295-3848 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (Grant Nos. 21427808, 21375077, and 21475076).
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DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.analchem.8b00831 Anal. Chem. XXXX, XXX, XXX−XXX
