Biosensors and Bioelectronics 53 (2014) 494–498
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Naked eye detection of trace cancer biomarkers based on biobarcode and enzyme-assisted DNA recycling hybrid amplifications Wenjiao Zhou, Jiao Su, Yaqin Chai, Ruo Yuan, Yun Xiang n Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 6 August 2013 Received in revised form 27 September 2013 Accepted 11 October 2013 Available online 24 October 2013
Naked eye-based detection has received increasing research interest due to the simplicity nature of this type of assay. However, improving the sensitivity of the naked eye detection method for the monitoring of trace amount of target molecules remains a major challenge. Herein, we describe a biobarcode and an enzyme-assisted DNA recycling hybrid amplification strategy for naked eye detection of sub-picomolar carcinoembryonic antigen (CEA), a cancer biomarker. The presence of CEA and the corresponding antibodies results in the formation of immunocomplexes and the capture of the biobarcodes in a microplate. The massive barcode DNAs released from the biobarcodes hybridize with the G-quadruplex inactive hairpin DNA probes and form catalytic nicking sites for N.BstNBI endonuclease, which cleaves the barcode DNA/hairpin partial dsDNA, releases the G-quadruplex active sequences and recycles the barcode DNA. Due to the barcode DNA recycling process, numerous G-quadruplex active sequences are generated and associate with hemin to form peroxidase mimicking enzymes, which convert colorless ABTS2 to green color intensified ABTS to achieve naked eye detection of CEA down to 0.025 ng mL 1 (0.14 pM). The naked eye detection strategy reported herein can be applied also to complicated serum sample matrix, making this approach hold great promise for point-of-care diagnostic applications. & 2013 Elsevier B.V. All rights reserved.
Keywords: Carcinoembryonic antigen Biobarcode amplification DNA recycling DNAzyme Visual detection
1. Introduction Cancer has become the number two leading causes of death in the US according to the report from National Cancer Institute. Cancer biomarkers (e.g., secreted proteins, DNA, mRNA and transcription factors) can distinguish normal or disease states (Wulfkuhle et al., 2003; Kulasingam and Diamandis, 2008; Rusling et al., 2010). The levels of cancer biomarkers can provide important information on the occurrence, existence and progression of different types of cancers (Hanash et al., 2008; Ludwig and Weinstein, 2005). Early identification and detection of these biomarkers are crucial for patient survival and successful prognosis of cancers (Giljohan and Mirkin, 2009; Wilson and Nock, 2003; Munge et al., 2011; Ferrari, 2005; Kingsmore, 2006). In this regard, intensive research focus has been directed toward developing robust analytical techniques for cancer biomarker detections. Indeed, in the past decades, we have witnessed the demonstrations of numerous methods for the detection of low levels of cancer biomarkers by coupling effective signal amplification schemes, such as immuno-PCR (Sano et al., 1992), rolling recycle
n
Corresponding author. Tel.: þ 86 23 68252277; fax: þ 86 23 68254000. E-mail address: [email protected] (Y. Xiang).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.020
amplification (Zhao et al., 2008; Nilsson et al., (2006); Kingsmore and Patel, 2003; Demidov, 2002), biobarcode (Nam et al., 2003, 2004; Stoeva et al., 2006a, b; Thaxton et al., 2009) and functional nanomaterials (Munge et al., 2011, 2005; Wang et al., 2004; Yu et al., 2006; Mani et al., 2009; Malhotra et al., 2010), with electrical, optical or mechanical transduction techniques. Despite the advantageous high sensitivity of these approaches, these methods are reliable only in laboratory settings and are not amenable for on-site or point-of-care (POC) applications due to the requirements of highly trained personnel and complicated signal transduction means. Therefore, the development of rapid, sensitive, selective and simple alternatives for cancer biomarker detection without using advanced or complicated instruments, for example by using human naked eye, can potentially revolutionize the detection and diagnosis of cancers. The naked eye-based detections were pioneered by Mirkin and colleagues (Mirkin et al., 1996; Storhoff et al., 1998), relying on the change of the optical properties of gold nanoparticles (AuNPs), which are strongly dependent upon the interparticle separation distance. The hybridizations between the target DNA and the DNA probes conjugated to AuNPs lead to the assembly of the AuNPs and cause a significant shift in the extinction spectrum of AuNPs, which is indicated by a visible color change from red to purple. Based on this type of target-induced assembly or disassembly of AuNPs detection mechanism, various visual sensing strategies
W. Zhou et al. / Biosensors and Bioelectronics 53 (2014) 494–498
have been developed to monitor different types of target molecules, including DNA (Li and Rothberg, 2004a, b; Du et al., 2006), small biomolecules (Liu and Lu, 2004, 2006; Wang et al., 2007) and metal ions (Choi et al., 2009; Darbha et al., 2008; Xu et al., 2010; He et al., 2008; Wang et al., 2008). Although these AuNP-based visual detection methods are simple, the AuNPs used in these techniques are susceptible to sensing environment (ionic strength, acidity, etc.), which may potentially lead to false positive responses (Liu and Lu, 2006; Laromaine et al., 2007). Besides, these visual detection approaches can be achieved with confidence only at high concentration of the target molecules (low sensitivity). Moreover, these strategies require the target molecules and the recognition probe molecules to be sufficiently small to guarantee a distinguishable color change upon assembly of AuNPs (Su et al., 2003). These limitations make sensitive naked eye detection of macromolecules (such as protein biomarkers) remain as a major challenge. To explore solutions for the challenges encountered in current naked eye-based detection of low levels of protein biomarkers, we propose herein a new strategy for sensitive and visual detection of carcinoembryonic antigen (CEA, a protein cancer biomarker) based on biobarcode and enzyme-assisted DNA recycling amplifications. Our approach employs the barcode DNA as the intermediate target, which can be recycled by an endonuclease to generate massive peroxidase mimicking enzymes, known as G-quadruplex/ hemin complexes. These mimicking enzymes catalyze the conversion of a colorless substrate to a green color product, which enable us to detect sub-picomolar CEA with naked eye.
2. Experimental section 2.1. Apparatus and reagents A 2450 UV spectrophotometer (Shimadzu, Japan) was used to obtain the absorption spectra at room temperature in all experiments. A canon EOS 550D camera was used to take all the photographs. All the DNA sequences (barcode DNA: 5′-TCATCACACTGGAAGACTC-3′; biotinylated barcode complementary DNA (c-DNA): 5′biotin-GAGTCTTCCAGTGTGATGA-3′; G-quadruplex inactive hairpin DNA: 5′-CCCTACCCGAGTCTTCCAGTGTGATGAGGGTAGGGCGGGTTGGG-3′) were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The nicking enzyme N. BstNBI (an endonuclease that recognizes the specific nucleotide sequence of 5′-GAGTC-3′ in a double-stranded DNA) and 10 NEBuffer 3 (50 mM Tris–HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, pH 7.9) were obtained from New England BioLabs (Ipswich, MA, USA). Hemin, 2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS), H2O2, and 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES) were purchased from Aladdin Reagents (Shanghai, China). The hemin stock solution (1 mM) was prepared in dimethylsulfoxide (DMSO) and stored at 20 1C. Streptavidin-coated magnetic microbeads (MyOne Streptavidin C1, STV-MBs) were obtained from Invitrogen Corp. (Oslo, Norway). The primary anti-CEA antibody (Ab1), biotinmodified secondary anti-CEA antibody (biotin-Ab2), CEA, alphafetoprotein (AFP), mouse IgG, vascular endothelial growth factor (VEGF) and the commercial CEA detection ELISA kit were all ordered from Biocell Co., Ltd (Zhengzhou, China). Human serum samples were provided by the 9th People's Hospital of Chongqing (Chongqing, China). 2.2. Preparation of the biobarcode conjugates An aliquot of 200 μL STV-MBs (10 mg mL 1) was washed twice with PBS buffer and incubated with PBS buffer containing biotinlylated Ab2 (2 μg mL 1) for 30 min at room temperature with
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gentle rotation. Afterwards, 30 μL of 100 μM c-DNA was added to conjugate with Ab2/STV-MB for 30 min at room temperature with gentle mixing. After washing the beads twice with PBS buffer and separating by an external magnet for 3 min, the beads were resuspended in PBS buffer. Finally, 30 μL of 100 μM barcode DNA was added and incubated with the c-DNA/Ab2/STV-MBs for 30 min. The resulting biobarcode conjugates were washed with PBS buffer and stored in PBS buffer at 4 1C. 2.3. Naked eye detection of CEA by using biobarcode and enzymeassisted barcode DNA recycling amplifications The polystyrene microplate (NUNC, Roskilde, Denmark) was first coated with Ab1 (10 μg mL 1, 100 μL, in 0.1 M carbonate buffer, pH 9.6) at 4 1C overnight. The wells were then washed with 200 μL PBST (0.01% Tween-20) three times and blocked with 200 μL PBS–BSA (1% BSA). Then, CEA solutions (100 μL) at different concentrations or serum samples were added to the wells and incubated at 37 1C for 1 h. After washing the wells with PBST three times, the biobarcode conjugate solution was incubated with the wells for 1 h at 37 1C, followed by washing twice with PBST. Subsequently, 100 μL of nanopure water was added to the wells, and the plate was heated to 60 1C for 5 min to ensure complete release of the barcode DNAs. The barcode DNA solution was separated by an external magnet and transferred into a 1.5 mL centrifuge tube containing 10 μL of 10 μM G-quadruplex inactive hairpin sequences, 15 μL 10 NEBuffer 3 and 30U N.BstNB I. The mixture was incubated at 55 1C for 60 min to allow recycling of the barcode DNA by the N.BstNB I enzyme (Lin et al., 2011). After the deactivation of the N.BstNB I enzyme by elevating the temperature to 90 1C, 5 μL of 1 μM hemin and 45 μL 2 HEPES buffer (50 mM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100 and 2% DMSO; pH 7.4) were added and incubated for 60 min at room temperature. Finally, ABTS2 and H2O2 were added to the mixture to attain final concentrations of 6 mM and 2 mM at room temperature, respectively, and photographs of the solutions were taken after 10 min of color development.
3. Results and discussion Our naked eye sensitive cancer biomarker detection principle is illustrated in Scheme 1. The biobarcode conjugates are first prepared by co-immobilizing biotin-Ab2 and biotin-c-DNA sequences on the streptavidin-coated magnetic microbeads (STVMBs) through strong biotin–STV affinity interactions. This is followed by the hybridizations between the barcode DNA and the corresponding complementary strands on the STV-MBs to obtain the biobarcode conjugates (Scheme 1A). In our biobarcode design, the 1 mm MBs, instead of the common 30 nm AuNPs, are used to prepare the biobarcodes due to the following considerations. First, by using the STV-MBs, the biobarcodes can be prepared within 1.5 h, which significantly reduces the biobarcode preparation time by avoiding the lengthy and complicated aging steps required in common AuNP-based biobarcode preparation processes. Second, due to the magnetic nature of the microbeads, the biobarcodes can be easily isolated by an external magnet and the 1 mm size of the beads can increase the loading of more barcode DNA as well. For naked eye detection (Scheme 1B), the target CEA molecules are incubated with Ab1-coated microplate, followed by the addition of the biobarcodes to form immuno-sandwich complexes in the wells of the microplate. After extensive washing to remove the non-specifically adsorbed biobarcodes, numerous barcode DNAs are released from the captured biobarcodes and transferred to a solution to hybridize with the G-quradruplex inactive hairpin DNA probes. These hybridizations
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in Fig. 1A(b), a clear green color change of ABTS2 is observed compared with the blank test (without CEA, Fig. 1A(a)). This color change is basically due to the barcode DNA-induced (hybridization between the barcode DNA and the hairpin probe) activation of the G-quadruplex sequences and formation of the mimicking enzyme,
Scheme 1. (A) Preparation of the biobarcode conjugate labels. The biotin-modified dsDNA containing the barcode DNA and the biotin–Ab2 are attached to the STVMBs through biotin/STV affinity interactions. (B) Principle of the naked eye sensitive detection of CEA based on biobarcode and enzyme-assisted DNA recycling amplifications. The presence of the target CEA leads to the formation of the sandwich immuno-complexes in the microplate, which is followed by the release of the barcode DNA and N.BstNBI enzyme-assisted barcode DNA recycling amplification for the generation of the G-quradruplex/hemin DNAzymes for producing the colored products for visual detection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
subsequently lead to unfolding of the hairpin structure and the formation of partial dsDNA, which creates specific restriction sites (5′ GAGTC 3′ in the dsDNA) for the N.BstNBI endonuclease. The N.BstNBI enzyme then cleaves these partial dsDNAs and releases the barcode DNA and the G-quadruplex active sequences. The released barcode DNAs again hybridize with the hairpin DNA probes and initiate the enzyme-assisted barcode DNA recycling process, which results in the generation of massive G-quadruplex active sequences. These G-quadruplex active sequences associate with hemin to form the peroxidase mimicking enzymes and catalyze the oxidation of the colorless ABTS2 to green color ABTS● in the presence of H2O2. Due to the dual amplification (biobarcode amplification and enzyme-assisted barcode DNA recycling) nature of the proposed strategy, the presence of low levels of CEA is expected to generate massive peroxidase mimicking enzymes and to induce a significant color change of ABTS2 for naked eye detection. For proof-of-concept demonstration, 40 ng mL 1 of CEA in buffer was first tested by our new strategy with and without the barcode DNA recycling amplification. After the formation of the immuno-complexes in the presence of CEA and the release of the barcode DNA, the barcode DNA was allowed to hybridize with the G-quradruplex inactive hairpin probes without the N.BstNBI enzyme, followed by the addition of hemin and ABTS2 . As shown
Fig. 1. (A) Photograph of naked eye detection of 0 ng mL 1 (a, blank) and 40 ng mL 1 CEA without (b) and with (c) barcode DNA recycling amplification. (B) The corresponding UV–vis absorption spectra of (A). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. (A) Photograph of the naked eye detection method for different concentrations of CEA: (a) 0, (b) 0.025, (c) 0.5, (d) 5, (e) 10, (f) 20 and (g) 40 ng mL 1. (B) UV– vis absorption spectra of a commercially available ELISA detection kit for CEA in the range of 0–40 ng mL 1. The absorbance spectra were recorded at 450 nm. Error bars: SD, n ¼3. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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Table 1 Comparisons between our proposed naked eye-based approach and some other typical reported methods for CEA detection. Reference Wei et al. (2011) Norouzi et al. (2011) Laboria et al. (2010) Su et al. (2011) Chen et al. (2011) This work
Detection method Colorimetry Square wave voltammetry Amperometry Voltammetry Chemiluminescence Naked eye detection
which leads to the oxidation of colorless ABTS2 to green color ABTS . However, with the addition of the N.BstNBI enzyme, which results in the recycling of the barcode DNA as discussed previously, the color change is drastically intensified (Fig. 1A(c)) at the same CEA level (40 ng mL 1). The color changes were monitored also by UV–vis spectra (Fig. 1B), in which the detection mode with barcode DNA amplification shows much stronger absorbance than that of the one without recycling amplification. These results shown here clearly demonstrate that the coupling of the enzyme-assisted DNA recycling with the barcode amplification can offer great potential for naked eye detection of trace CEA. To further evaluate whether the color signal change is dependent upon the concentration of CEA, different concentrations of CEA ranging from 0.025 to 40 ng mL 1 were tested with our new method. As displayed in Fig. 2A, we can see that with the presence of increasing concentration of CEA, the color signal output is gradually intensified (a-g), and the presence of 0.5 ng mL 1 CEA leads to an unambiguous color change compared with the blank test (0 ng mL 1 CEA). Moreover, the presence of even as low as 0.025 ng mL 1 (0.14 pM) CEA can be distinguished from the blank test. Furthermore, the detection limit of our naked eye-based approach for CEA is comparable with those of some other reported methods based on optical or electrochemical amplification labels (Table 1). Considering the threshold concentration of CEA for healthy person (2.5 ng mL 1) and smokers (5.0 ng mL 1), our developed method is sensitive enough for early diagnosis of CEA without using any advanced instrument. A commercial colorimetric CEA detection kit was also employed for direct analytical performance comparison. The commercial CEA detection kit uses a conventional enzyme-linked immunosorbent assay (ELISA) approach, in which the presence of the target CEA results in the formation of Ab1/CEA/Ab2–horseradish peroxidase (HRP) immunocomplexes. The captured HRP catalyzes the conversion of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to a colorful product, which can be monitored with UV–vis at 450 nm. In the investigated concentration range of CEA (0–40 ng mL 1), the absorbance intensity exhibits a dynamic relationship with the concentration of CEA, and 1.0 ng mL 1 of CEA can be detected (Fig. 2B). This direct comparison indicates that our method shows significant improvement (40-fold) in the detection limit (0.025 vs 1 ng mL 1) over the commercial detection kit, which is basically owing to the two amplification steps involved in our new assay protocol. To investigate the specificity and selectivity of our visual detection strategy for CEA, three other non-target proteins, AFP, mouse IgG and VEGF, were tested as control molecules. According to the photographic results in Fig. 3, the presence of even 5-fold excess of the non-target proteins (100 vs 20 ng mL 1) causes minimal color change compared with that of the blank experiment. However, the presence of a low concentration of CEA (20 ng mL 1) yields a remarkable color signal. These results reveal that the sensing performance of our visual method is not significantly affected by other non-target analytes. In other words, the target CEA can be selectively detected in a visible fashion by naked eye.
Detection limit 1
0.25 μg mL 0.01 ng mL 1 0.2 ng mL 1 0.005 ng mL 1 0.1 ng mL 1 0.025 ng mL 1
Signal amplification strategy Direct detection method Au/ZnO NPs bilayer film Bipodal thiolated self-assembled monolayer Multiarmed star-like Pt nanowires Functionalized mesoporous silica nanoparticles Biobarcode and niking enzyme dual amplifications
Fig. 3. Selectivity evaluation of the proposed method for CEA against other nontarget proteins: (a) blank, (b) VEGF 100 ng mL 1, (c) mouse IgG 100 ng mL 1, (d) AFP 100 ng mL 1 and (e) CEA 20 ng mL 1. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 4. Photograph of the naked eye detection method for (a) blank test (0 ng mL 1 in buffer) and different concentrations of CEA spiked in 10% human serum: (b) 0, (c) 0.025, (d) 5, (e) 10, (f) 20 and (g) 40 ng mL 1. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
The CEA recognition in our experimental design involves the use of an ELISA format, which holds potential application for clinical analyses. To apply our visual detection method to serum samples, 10% human serum samples spiked with/without CEA were examined. As can be seen in Fig. 4, the serum sample without spiked CEA shows no obvious color change compared with that in the blank test (Fig. 4b vs a), while the serum sample with the spiked CEA concentration higher than 5 ng mL 1 (Fig. 4d) exhibits clear green color signal, indicating that our method can be applied to complex serum media.
4. Conclusions In conclusion, we have demonstrated a naked eye detection method for sensitive monitoring of cancer biomarker CEA. By coupling the biobarcode amplification with enzyme-assisted DNA recycling, we can visually detect CEA down to 0.025 ng mL 1 without using complicated instruments. Our developed method shows high selectivity against non-target proteins and can be applied to serum samples. The naked eye-based detection strategy proved herein may thus serve as a useful alternative to current visual assays with complicated procedures and poor detection limits. Although we demonstrated the detection of only CEA in current work, this methodology can be easily expanded for the detection of more general targets by coupling with different biorecognition events (e.g., DNA hybridization, sugar/lectin interaction and aptamer/target binding).
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Acknowledgment This work was supported by NSFC (Nos. 21275004, 20905062, 21075100 and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932), and Fundamental Research Funds for the Central Universities (XDJK2012A004).
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Naked eye detection of trace cancer biomarkers based on biobarcode and enzyme-assisted DNA recycling hybrid amplifications Wenjiao Zhou, Jiao Su, Yaqin Chai, Ruo Yuan, Yun Xiang n Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 6 August 2013 Received in revised form 27 September 2013 Accepted 11 October 2013 Available online 24 October 2013
Naked eye-based detection has received increasing research interest due to the simplicity nature of this type of assay. However, improving the sensitivity of the naked eye detection method for the monitoring of trace amount of target molecules remains a major challenge. Herein, we describe a biobarcode and an enzyme-assisted DNA recycling hybrid amplification strategy for naked eye detection of sub-picomolar carcinoembryonic antigen (CEA), a cancer biomarker. The presence of CEA and the corresponding antibodies results in the formation of immunocomplexes and the capture of the biobarcodes in a microplate. The massive barcode DNAs released from the biobarcodes hybridize with the G-quadruplex inactive hairpin DNA probes and form catalytic nicking sites for N.BstNBI endonuclease, which cleaves the barcode DNA/hairpin partial dsDNA, releases the G-quadruplex active sequences and recycles the barcode DNA. Due to the barcode DNA recycling process, numerous G-quadruplex active sequences are generated and associate with hemin to form peroxidase mimicking enzymes, which convert colorless ABTS2 to green color intensified ABTS to achieve naked eye detection of CEA down to 0.025 ng mL 1 (0.14 pM). The naked eye detection strategy reported herein can be applied also to complicated serum sample matrix, making this approach hold great promise for point-of-care diagnostic applications. & 2013 Elsevier B.V. All rights reserved.
Keywords: Carcinoembryonic antigen Biobarcode amplification DNA recycling DNAzyme Visual detection
1. Introduction Cancer has become the number two leading causes of death in the US according to the report from National Cancer Institute. Cancer biomarkers (e.g., secreted proteins, DNA, mRNA and transcription factors) can distinguish normal or disease states (Wulfkuhle et al., 2003; Kulasingam and Diamandis, 2008; Rusling et al., 2010). The levels of cancer biomarkers can provide important information on the occurrence, existence and progression of different types of cancers (Hanash et al., 2008; Ludwig and Weinstein, 2005). Early identification and detection of these biomarkers are crucial for patient survival and successful prognosis of cancers (Giljohan and Mirkin, 2009; Wilson and Nock, 2003; Munge et al., 2011; Ferrari, 2005; Kingsmore, 2006). In this regard, intensive research focus has been directed toward developing robust analytical techniques for cancer biomarker detections. Indeed, in the past decades, we have witnessed the demonstrations of numerous methods for the detection of low levels of cancer biomarkers by coupling effective signal amplification schemes, such as immuno-PCR (Sano et al., 1992), rolling recycle
n
Corresponding author. Tel.: þ 86 23 68252277; fax: þ 86 23 68254000. E-mail address: [email protected] (Y. Xiang).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.020
amplification (Zhao et al., 2008; Nilsson et al., (2006); Kingsmore and Patel, 2003; Demidov, 2002), biobarcode (Nam et al., 2003, 2004; Stoeva et al., 2006a, b; Thaxton et al., 2009) and functional nanomaterials (Munge et al., 2011, 2005; Wang et al., 2004; Yu et al., 2006; Mani et al., 2009; Malhotra et al., 2010), with electrical, optical or mechanical transduction techniques. Despite the advantageous high sensitivity of these approaches, these methods are reliable only in laboratory settings and are not amenable for on-site or point-of-care (POC) applications due to the requirements of highly trained personnel and complicated signal transduction means. Therefore, the development of rapid, sensitive, selective and simple alternatives for cancer biomarker detection without using advanced or complicated instruments, for example by using human naked eye, can potentially revolutionize the detection and diagnosis of cancers. The naked eye-based detections were pioneered by Mirkin and colleagues (Mirkin et al., 1996; Storhoff et al., 1998), relying on the change of the optical properties of gold nanoparticles (AuNPs), which are strongly dependent upon the interparticle separation distance. The hybridizations between the target DNA and the DNA probes conjugated to AuNPs lead to the assembly of the AuNPs and cause a significant shift in the extinction spectrum of AuNPs, which is indicated by a visible color change from red to purple. Based on this type of target-induced assembly or disassembly of AuNPs detection mechanism, various visual sensing strategies
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have been developed to monitor different types of target molecules, including DNA (Li and Rothberg, 2004a, b; Du et al., 2006), small biomolecules (Liu and Lu, 2004, 2006; Wang et al., 2007) and metal ions (Choi et al., 2009; Darbha et al., 2008; Xu et al., 2010; He et al., 2008; Wang et al., 2008). Although these AuNP-based visual detection methods are simple, the AuNPs used in these techniques are susceptible to sensing environment (ionic strength, acidity, etc.), which may potentially lead to false positive responses (Liu and Lu, 2006; Laromaine et al., 2007). Besides, these visual detection approaches can be achieved with confidence only at high concentration of the target molecules (low sensitivity). Moreover, these strategies require the target molecules and the recognition probe molecules to be sufficiently small to guarantee a distinguishable color change upon assembly of AuNPs (Su et al., 2003). These limitations make sensitive naked eye detection of macromolecules (such as protein biomarkers) remain as a major challenge. To explore solutions for the challenges encountered in current naked eye-based detection of low levels of protein biomarkers, we propose herein a new strategy for sensitive and visual detection of carcinoembryonic antigen (CEA, a protein cancer biomarker) based on biobarcode and enzyme-assisted DNA recycling amplifications. Our approach employs the barcode DNA as the intermediate target, which can be recycled by an endonuclease to generate massive peroxidase mimicking enzymes, known as G-quadruplex/ hemin complexes. These mimicking enzymes catalyze the conversion of a colorless substrate to a green color product, which enable us to detect sub-picomolar CEA with naked eye.
2. Experimental section 2.1. Apparatus and reagents A 2450 UV spectrophotometer (Shimadzu, Japan) was used to obtain the absorption spectra at room temperature in all experiments. A canon EOS 550D camera was used to take all the photographs. All the DNA sequences (barcode DNA: 5′-TCATCACACTGGAAGACTC-3′; biotinylated barcode complementary DNA (c-DNA): 5′biotin-GAGTCTTCCAGTGTGATGA-3′; G-quadruplex inactive hairpin DNA: 5′-CCCTACCCGAGTCTTCCAGTGTGATGAGGGTAGGGCGGGTTGGG-3′) were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The nicking enzyme N. BstNBI (an endonuclease that recognizes the specific nucleotide sequence of 5′-GAGTC-3′ in a double-stranded DNA) and 10 NEBuffer 3 (50 mM Tris–HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, pH 7.9) were obtained from New England BioLabs (Ipswich, MA, USA). Hemin, 2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS), H2O2, and 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES) were purchased from Aladdin Reagents (Shanghai, China). The hemin stock solution (1 mM) was prepared in dimethylsulfoxide (DMSO) and stored at 20 1C. Streptavidin-coated magnetic microbeads (MyOne Streptavidin C1, STV-MBs) were obtained from Invitrogen Corp. (Oslo, Norway). The primary anti-CEA antibody (Ab1), biotinmodified secondary anti-CEA antibody (biotin-Ab2), CEA, alphafetoprotein (AFP), mouse IgG, vascular endothelial growth factor (VEGF) and the commercial CEA detection ELISA kit were all ordered from Biocell Co., Ltd (Zhengzhou, China). Human serum samples were provided by the 9th People's Hospital of Chongqing (Chongqing, China). 2.2. Preparation of the biobarcode conjugates An aliquot of 200 μL STV-MBs (10 mg mL 1) was washed twice with PBS buffer and incubated with PBS buffer containing biotinlylated Ab2 (2 μg mL 1) for 30 min at room temperature with
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gentle rotation. Afterwards, 30 μL of 100 μM c-DNA was added to conjugate with Ab2/STV-MB for 30 min at room temperature with gentle mixing. After washing the beads twice with PBS buffer and separating by an external magnet for 3 min, the beads were resuspended in PBS buffer. Finally, 30 μL of 100 μM barcode DNA was added and incubated with the c-DNA/Ab2/STV-MBs for 30 min. The resulting biobarcode conjugates were washed with PBS buffer and stored in PBS buffer at 4 1C. 2.3. Naked eye detection of CEA by using biobarcode and enzymeassisted barcode DNA recycling amplifications The polystyrene microplate (NUNC, Roskilde, Denmark) was first coated with Ab1 (10 μg mL 1, 100 μL, in 0.1 M carbonate buffer, pH 9.6) at 4 1C overnight. The wells were then washed with 200 μL PBST (0.01% Tween-20) three times and blocked with 200 μL PBS–BSA (1% BSA). Then, CEA solutions (100 μL) at different concentrations or serum samples were added to the wells and incubated at 37 1C for 1 h. After washing the wells with PBST three times, the biobarcode conjugate solution was incubated with the wells for 1 h at 37 1C, followed by washing twice with PBST. Subsequently, 100 μL of nanopure water was added to the wells, and the plate was heated to 60 1C for 5 min to ensure complete release of the barcode DNAs. The barcode DNA solution was separated by an external magnet and transferred into a 1.5 mL centrifuge tube containing 10 μL of 10 μM G-quadruplex inactive hairpin sequences, 15 μL 10 NEBuffer 3 and 30U N.BstNB I. The mixture was incubated at 55 1C for 60 min to allow recycling of the barcode DNA by the N.BstNB I enzyme (Lin et al., 2011). After the deactivation of the N.BstNB I enzyme by elevating the temperature to 90 1C, 5 μL of 1 μM hemin and 45 μL 2 HEPES buffer (50 mM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100 and 2% DMSO; pH 7.4) were added and incubated for 60 min at room temperature. Finally, ABTS2 and H2O2 were added to the mixture to attain final concentrations of 6 mM and 2 mM at room temperature, respectively, and photographs of the solutions were taken after 10 min of color development.
3. Results and discussion Our naked eye sensitive cancer biomarker detection principle is illustrated in Scheme 1. The biobarcode conjugates are first prepared by co-immobilizing biotin-Ab2 and biotin-c-DNA sequences on the streptavidin-coated magnetic microbeads (STVMBs) through strong biotin–STV affinity interactions. This is followed by the hybridizations between the barcode DNA and the corresponding complementary strands on the STV-MBs to obtain the biobarcode conjugates (Scheme 1A). In our biobarcode design, the 1 mm MBs, instead of the common 30 nm AuNPs, are used to prepare the biobarcodes due to the following considerations. First, by using the STV-MBs, the biobarcodes can be prepared within 1.5 h, which significantly reduces the biobarcode preparation time by avoiding the lengthy and complicated aging steps required in common AuNP-based biobarcode preparation processes. Second, due to the magnetic nature of the microbeads, the biobarcodes can be easily isolated by an external magnet and the 1 mm size of the beads can increase the loading of more barcode DNA as well. For naked eye detection (Scheme 1B), the target CEA molecules are incubated with Ab1-coated microplate, followed by the addition of the biobarcodes to form immuno-sandwich complexes in the wells of the microplate. After extensive washing to remove the non-specifically adsorbed biobarcodes, numerous barcode DNAs are released from the captured biobarcodes and transferred to a solution to hybridize with the G-quradruplex inactive hairpin DNA probes. These hybridizations
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in Fig. 1A(b), a clear green color change of ABTS2 is observed compared with the blank test (without CEA, Fig. 1A(a)). This color change is basically due to the barcode DNA-induced (hybridization between the barcode DNA and the hairpin probe) activation of the G-quadruplex sequences and formation of the mimicking enzyme,
Scheme 1. (A) Preparation of the biobarcode conjugate labels. The biotin-modified dsDNA containing the barcode DNA and the biotin–Ab2 are attached to the STVMBs through biotin/STV affinity interactions. (B) Principle of the naked eye sensitive detection of CEA based on biobarcode and enzyme-assisted DNA recycling amplifications. The presence of the target CEA leads to the formation of the sandwich immuno-complexes in the microplate, which is followed by the release of the barcode DNA and N.BstNBI enzyme-assisted barcode DNA recycling amplification for the generation of the G-quradruplex/hemin DNAzymes for producing the colored products for visual detection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
subsequently lead to unfolding of the hairpin structure and the formation of partial dsDNA, which creates specific restriction sites (5′ GAGTC 3′ in the dsDNA) for the N.BstNBI endonuclease. The N.BstNBI enzyme then cleaves these partial dsDNAs and releases the barcode DNA and the G-quadruplex active sequences. The released barcode DNAs again hybridize with the hairpin DNA probes and initiate the enzyme-assisted barcode DNA recycling process, which results in the generation of massive G-quadruplex active sequences. These G-quadruplex active sequences associate with hemin to form the peroxidase mimicking enzymes and catalyze the oxidation of the colorless ABTS2 to green color ABTS● in the presence of H2O2. Due to the dual amplification (biobarcode amplification and enzyme-assisted barcode DNA recycling) nature of the proposed strategy, the presence of low levels of CEA is expected to generate massive peroxidase mimicking enzymes and to induce a significant color change of ABTS2 for naked eye detection. For proof-of-concept demonstration, 40 ng mL 1 of CEA in buffer was first tested by our new strategy with and without the barcode DNA recycling amplification. After the formation of the immuno-complexes in the presence of CEA and the release of the barcode DNA, the barcode DNA was allowed to hybridize with the G-quradruplex inactive hairpin probes without the N.BstNBI enzyme, followed by the addition of hemin and ABTS2 . As shown
Fig. 1. (A) Photograph of naked eye detection of 0 ng mL 1 (a, blank) and 40 ng mL 1 CEA without (b) and with (c) barcode DNA recycling amplification. (B) The corresponding UV–vis absorption spectra of (A). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. (A) Photograph of the naked eye detection method for different concentrations of CEA: (a) 0, (b) 0.025, (c) 0.5, (d) 5, (e) 10, (f) 20 and (g) 40 ng mL 1. (B) UV– vis absorption spectra of a commercially available ELISA detection kit for CEA in the range of 0–40 ng mL 1. The absorbance spectra were recorded at 450 nm. Error bars: SD, n ¼3. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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Table 1 Comparisons between our proposed naked eye-based approach and some other typical reported methods for CEA detection. Reference Wei et al. (2011) Norouzi et al. (2011) Laboria et al. (2010) Su et al. (2011) Chen et al. (2011) This work
Detection method Colorimetry Square wave voltammetry Amperometry Voltammetry Chemiluminescence Naked eye detection
which leads to the oxidation of colorless ABTS2 to green color ABTS . However, with the addition of the N.BstNBI enzyme, which results in the recycling of the barcode DNA as discussed previously, the color change is drastically intensified (Fig. 1A(c)) at the same CEA level (40 ng mL 1). The color changes were monitored also by UV–vis spectra (Fig. 1B), in which the detection mode with barcode DNA amplification shows much stronger absorbance than that of the one without recycling amplification. These results shown here clearly demonstrate that the coupling of the enzyme-assisted DNA recycling with the barcode amplification can offer great potential for naked eye detection of trace CEA. To further evaluate whether the color signal change is dependent upon the concentration of CEA, different concentrations of CEA ranging from 0.025 to 40 ng mL 1 were tested with our new method. As displayed in Fig. 2A, we can see that with the presence of increasing concentration of CEA, the color signal output is gradually intensified (a-g), and the presence of 0.5 ng mL 1 CEA leads to an unambiguous color change compared with the blank test (0 ng mL 1 CEA). Moreover, the presence of even as low as 0.025 ng mL 1 (0.14 pM) CEA can be distinguished from the blank test. Furthermore, the detection limit of our naked eye-based approach for CEA is comparable with those of some other reported methods based on optical or electrochemical amplification labels (Table 1). Considering the threshold concentration of CEA for healthy person (2.5 ng mL 1) and smokers (5.0 ng mL 1), our developed method is sensitive enough for early diagnosis of CEA without using any advanced instrument. A commercial colorimetric CEA detection kit was also employed for direct analytical performance comparison. The commercial CEA detection kit uses a conventional enzyme-linked immunosorbent assay (ELISA) approach, in which the presence of the target CEA results in the formation of Ab1/CEA/Ab2–horseradish peroxidase (HRP) immunocomplexes. The captured HRP catalyzes the conversion of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to a colorful product, which can be monitored with UV–vis at 450 nm. In the investigated concentration range of CEA (0–40 ng mL 1), the absorbance intensity exhibits a dynamic relationship with the concentration of CEA, and 1.0 ng mL 1 of CEA can be detected (Fig. 2B). This direct comparison indicates that our method shows significant improvement (40-fold) in the detection limit (0.025 vs 1 ng mL 1) over the commercial detection kit, which is basically owing to the two amplification steps involved in our new assay protocol. To investigate the specificity and selectivity of our visual detection strategy for CEA, three other non-target proteins, AFP, mouse IgG and VEGF, were tested as control molecules. According to the photographic results in Fig. 3, the presence of even 5-fold excess of the non-target proteins (100 vs 20 ng mL 1) causes minimal color change compared with that of the blank experiment. However, the presence of a low concentration of CEA (20 ng mL 1) yields a remarkable color signal. These results reveal that the sensing performance of our visual method is not significantly affected by other non-target analytes. In other words, the target CEA can be selectively detected in a visible fashion by naked eye.
Detection limit 1
0.25 μg mL 0.01 ng mL 1 0.2 ng mL 1 0.005 ng mL 1 0.1 ng mL 1 0.025 ng mL 1
Signal amplification strategy Direct detection method Au/ZnO NPs bilayer film Bipodal thiolated self-assembled monolayer Multiarmed star-like Pt nanowires Functionalized mesoporous silica nanoparticles Biobarcode and niking enzyme dual amplifications
Fig. 3. Selectivity evaluation of the proposed method for CEA against other nontarget proteins: (a) blank, (b) VEGF 100 ng mL 1, (c) mouse IgG 100 ng mL 1, (d) AFP 100 ng mL 1 and (e) CEA 20 ng mL 1. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
Fig. 4. Photograph of the naked eye detection method for (a) blank test (0 ng mL 1 in buffer) and different concentrations of CEA spiked in 10% human serum: (b) 0, (c) 0.025, (d) 5, (e) 10, (f) 20 and (g) 40 ng mL 1. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
The CEA recognition in our experimental design involves the use of an ELISA format, which holds potential application for clinical analyses. To apply our visual detection method to serum samples, 10% human serum samples spiked with/without CEA were examined. As can be seen in Fig. 4, the serum sample without spiked CEA shows no obvious color change compared with that in the blank test (Fig. 4b vs a), while the serum sample with the spiked CEA concentration higher than 5 ng mL 1 (Fig. 4d) exhibits clear green color signal, indicating that our method can be applied to complex serum media.
4. Conclusions In conclusion, we have demonstrated a naked eye detection method for sensitive monitoring of cancer biomarker CEA. By coupling the biobarcode amplification with enzyme-assisted DNA recycling, we can visually detect CEA down to 0.025 ng mL 1 without using complicated instruments. Our developed method shows high selectivity against non-target proteins and can be applied to serum samples. The naked eye-based detection strategy proved herein may thus serve as a useful alternative to current visual assays with complicated procedures and poor detection limits. Although we demonstrated the detection of only CEA in current work, this methodology can be easily expanded for the detection of more general targets by coupling with different biorecognition events (e.g., DNA hybridization, sugar/lectin interaction and aptamer/target binding).
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Acknowledgment This work was supported by NSFC (Nos. 21275004, 20905062, 21075100 and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932), and Fundamental Research Funds for the Central Universities (XDJK2012A004).
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