All-in-one dual-aptasensor capable of rapidly quantifying carcinoembryonic antigen

Highlights

• Design of dual DNA aptamers capable of rapidly capturing CEA and hemin.

• Competition of CEA and hemin to bind with the dual DNA aptamer in human serum.

• All-in-one dual-aptasemsor devised with the combination of CEA, hemin and dual DNA aptamer.

• Rapid quantification of CEA using the all-in-one dual-aptasensor with good reproducibility.

Abstract

Using a dual DNA aptamer (CEA aptamer linked to hemin aptamer), capable of rapidly capturing carcinoembryonic antigen (CEA) and hemin, an all-in-one dual-aptasensor with 1,1′-oxalyldiimidazole (ODI) chemiluminescence detection was developed for the early diagnosis of human cancer. CEA and hemin competitively bound with the dual DNA aptamer while the mixture in a detection cell was incubated for 30 min at room temperature. When Amplex Red and H₂O₂ were added in the detection cell after the incubation, the yield of resorufin formed from the reaction Amplex Red and H₂O₂ depended on the concentration of HRP-mimicking G-quardruplex DNAzyme formed from the binding interaction between hemin and the dual DNA aptamer. Bright red light was observed with the addition of ODI and H₂O₂ in the detection cell containing resorufin. Relative CL intensity of all-in-one dual-aptasensor, operated with the competitive reaction of CEA and hemin in the presence of the dual aptamer, was exponentially decreased with the increase of CEA concentration in human serum. The limit of detection (LOD=3σ) of the all-in-one dual-aptasensor which operated with excellent accuracy, precision, and reproducibility was as low as 0.58 ng/ml. The good correlation between the easy to use all-in-one dual-aptasensor and conventional enzyme-linked immunosorbent assay (ELISA), operated with time consuming procedures (e.g., long incubations and multiple washings), indicates that the rapid all-in-one dual-aptasensor can be applied as a novel clinical tool for the early diagnosis of breast cancer.

Introduction

Recently, a number of dual-aptasensors for the early diagnosis and rapid prognosis of human diseases (e.g., acute myocardial infarction, cancer, infectious diseases) have been developed (Arif et al., 2015, Kadioglu et al., 2015, Kirsch et al., 2013, Ping et al., 2015). This is because various fatal human diseases can often be treated if they are diagnosed early.

In order to early diagnose human diseases, most of dual-aptasensors have generally been developed based on the principle of immunoassay operated with specific antibodies (e.g., capture and detection antibodies) capable of rapidly binding a biomarker existing in human samples (Camey et al., 2003, Diaconu et al., 2013, Pei et al., 2013). A detection antibody conjugated with a specific enzyme such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP) is widely used to enhance the sensitivity of dual-aptasensor. Sandwich enzyme immunoassay with a capture antibody and detection antibody-conjugated HRP or ALP is highly sensitive with acceptable accuracy and reproducibility (Chong et al., 2012, Kim et al., 2014, Lang et al., 2014). However, antibodies and enzymes obtained from the sacrifice of animals are very expensive and intractable in ambient condition.

Many research groups have developed various methods to immobilize multiple detection antibodies and enzymes on the surface of nanoparticles such as gold, platinum, and silver (Choi et al., 2017, Wang et al., 2014a, Wang et al., 2014b) Using the nanoparticles modified with detection antibody-conjugated HRP, it was possible to develop more sensitive enzyme immunoassays.(Huang and Ren, 2011; Liang et al., 2015) However, the system of dual-aptasensor was more expensive and more complicated than conventional enzyme immunoassay.

Since 1990, various aptamers composed of DNA, RNA, or peptide have been discovered and designed with the systematic evolution of ligands by exponential enrichment (SELEX) (Bock et al., 1992; Toh et al., 2015). This is because the binding rate of a specific aptamer to capture a biomarker in human sample is similar to or more rapid than that of a conventional antibody (Bock et al., 1992, Toh et al., 2015).

Using the advantages of aptamer, many dual-aptasensors were developed as a medical device because the selectivity of dual-aptasensor using DNA or RNA aptamer conjugated with fluorescence dye (e.g., fluorescein, 6-FAM) are acceptable for the early diagnosis of human diseases (Cha et al., 2014, Cho et al., 2014). Unfortunately, the sensitivity of aptasensor using a detection aptamer-conjugated fluorescent dye was not as good as that of dual-aptasensor using a detection antibody conjugated with HRP. In order to solve the disadvantages of aptasensor, recently, various nanoparticles as well as microspheres (e.g., magnetic beads) were used to enhance the sensitivity of aptasensor (Shi et al., 2014, Soh et al., 2015, Yang et al., 2015). Consequently, the complicated aptasensors, like enzyme immunoassays, were operated with time-consuming procedures such as multiple incubations and washings.

Hemin shown in Fig. S1(A) is well-known as a HRP-mimicking compound. Also, the efficiency of hemin bound with DNA hemin aptamers, called HRP-mimicking G-quadruplex DNAzyme shown in Fig. S1(B), is as good as that of HRP in enzyme immunoassay (Whillner et al., 2008). As the additional advantage of the HRP-mimicking G-quadruplex DNAzyme, it is stable at ambient condition. Thus, several aptasensors using aptamers and HRP-mimicking G-quadruplex DNAzyme have been developed as diagnostic methods (Freeman et al., 2012, Niazov-Elkan et al., 2014). The aptasensor using HRP-mimicking G-quadruplex DNAzyme was more sensitive than the aptasensor using fluorescent dye even though the operation method of the former was more complicated than that of the latter. This is because the aptasensor using HRP-mimicking G-quadruplex DNAzyme is operated with time-consuming procedures such as multiple incubations and multiple washings.

Colorimetric, fluorescence, and chemiluminescence are widely applied as optical detection methods of conventional immunoassays (Beloglazova and Eremin, 2015, Lee et al., 2010, Zhan et al., 2014) as well as aptasensors (Choi and Lee, 2013, Pang et al., 2015, Ramezani et al., 2015). Chemiluminescence detection is more sensitive than other optical sensors because the background of chemiluminescence emitted from a chemical reaction is lower than those of absorbance and fluorescence generated by a light source (Park et al., 2013). Recently, it was confirmed that the enzyme immunoassay with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) is more sensitive than that with luminol chemiluminescence (Lee et al., 2010). The previous report implies that ODI-CL as a detection method of the aptasensor using G-quadruplex DNAzyme is more sensitive than the luminol chemiluminescence, which is widely applied as a detection method of the aptasensor with G-quadruplex DNAzyme (Gribas et al., 2015, Jiang et al., 2013a, Zhou et al., 2012). ODI-CL has yet to be used as a detection method of the aptasensor using G-quadruplex DNAzyme. As shown in Fig. S1(C), however, the plausible reaction mechanism of ODI-CL reaction in the presence of G-quadruplex DNAzyme, instead of HRP, can be proposed.

Carcinoembryonic antigen (CEA) is well-known as a cancer marker for the diagnoses of various human cancers such as breast, Currently, sandwich enzyme immunoassay using a detection antibody conjugated with HRP or ALP is widely applied (Kim et al., 2014, Yang et al., 2009, Yang et al., 2010). Recently, two different types of DNA aptamers, capable of rapidly and selectively capturing CEA in a sample, have been developed (Shu et al., 2013).

Recently, dual aptamers, composed of two different aptamers combined with a specific linker, were able to quantify trace levels of toxins in a sample (Jo et al., 2016, Mun et al., 2014). It is possible that each aptamer of the dual aptamer was able to bind independently with a specific marker in a sample. In this research, we designed dual DNA aptamers (e.g., 5′-CEA aptamer-linker-hemin aptamer-3′, 5′-hemin aptamer-linker-CEA aptamer-3′) using the combination of CEA aptamer, hemin aptamer and a linker composed of 5 adenines. It is possible that the dual DNA aptamer can bind with CEA in human serum as well as hemin, to form HRP-mimicking G-quadruplex DNAzyme. Using the dual DNA aptamer designed based on the hypothesis, we developed for the first time an easy to use all-in-one dual-aptasensor with ODI chemiluminescence detection capable of rapidly quantifying CEA without time consuming and tedious multiple washings after incubation. Details are reported below.