A single quantum dot-based biosensor for telomerase assayby Guichi Zhu, Kun Yang, Chun-yang Zhang

Chem. Commun.

Similar

Enzyme biosensor based on NAD-sensitive quantum dots

Authors:
Xiangling Ren, Liuqing Yang, Fangqiong Tang, Chuanmiao Yan, Jun Ren
2010

FRET-Based Biosensor for Oleic Acid in Nanomolar Range with Quantum Dots As an Energy Donor

Authors:
Sergey V. Dezhurov, Irina Y. Volkova, Maxim S. Wakstein
2011

Single white light emitting hybrid nanoarchitectures based on functionalized quantum dots

Authors:
Elisabetta Fanizza, Carmine Urso, Vita Pinto, Antonio Cardone, Roberta Ragni, Nicoletta Depalo, M. Lucia Curri, Angela Agostiano, Gianluca M. Farinola, Marinella Striccoli
2014

Text

This journal is©The Royal Society of Chemistry 2015 Chem. Commun.

Cite this:DOI: 10.1039/c5cc01600b

A single quantum dot-based biosensor for telomerase assay†

Guichi Zhu, Kun Yang and Chun-yang Zhang*

Telomerase is a promising biomarker and a therapeutic target due to its extensive expression in human tumors such as lung cancer and breast cancer. Here, we develop a single quantum dot (QD)-based biosensor for the sensitive detection of telomerase activity. This single

QD-based biosensor has significant advantages of simplicity and high sensitivity, and it can be applied for the discrimination of tumor cells from normal cells as well as the screening of anticancer drugs.

Human telomerase is a reverse transcriptase with a special complex structure including a functional protein component and an endogenous RNA template, and it can specifically add several repeat units to the ends of a telomere.1 In normal cells, the expression of telomerase is usually repressed, and the telomeres shorten progressively with each cell division, resulting in cell senescence and death.2 However, in human cancer cells, the length of telomeres is maintained due to either the up-regulation or the reactivation of telomerase activity, making cancer cells divide indefinitely.3 Especially, a majority of cancers demonstrate positive telomerase activity.4 Therefore, the measurement of telomerase activity is essential for both clinical diagnosis and anticancer-drug discovery.

Since the discovery of telomerase in 1985,5 a variety of methods have been developed for the telomerase assay. Among them, the radioactive approach enables the direct measurement of telomerase activity, but it suffers from poor sensitivity and radiation risk.6

To improve the detection sensitivity, a polymerase chain reaction (PCR)-based telomere repeat amplification protocol (TRAP) is introduced7 and it can measure telomerase at the single-cell level,8 but it involves the precise control of temperature cycling and cannot detect very short telomerase products.9 So far, the reported alternative methods for the telomerase assay include immunoassay,10 the molecular beacon-based assay,11 the isothermal amplification reaction-based method,12 the electrochemical assay,13 and the gold nanoparticle-based method.14 However, the requirement of expensive antibodies10 and doubly labeled fluorescent probes,11 the involvement of various enzymes12 and the immobilization/ separation steps,13 and the time-consuming synthesis of gold nanoparticles14 might hinder their practical applications. Thus the development of a simple and sensitive method for the telomerase assay still remains a great challenge.

Semiconductor quantum dots (QDs) exhibit unique optical properties including high quantum yield, good stability against photobleaching, narrow luminescence bands and size-tunable luminescence spectra, and have been widely applied in fluorescence imaging, drug delivery and biomedical research.15 Especially, the QDs can be employed for the development of fluorescence resonance energy transfer (FRET)-based sensors for the detection of nucleic acids, proteins, and even small molecules.16 Here, we develop a single QD-based biosensor for sensitive detection of telomerase activity. In the presence of telomerase, Cy5 molecules can be assembled in an orderly manner into the ends of primer with the assembly direction towards the QD, enabling the enhanced FRET efficiency between the QD and Cy5. However, in the absence of telomerase, no Cy5 molecule can be assembled onto the surface of QD and consequently no FRET occurs between the

QD and Cy5, resulting in a near zero-background signal. This single

QD-based biosensor has the significant advantages of simplicity and high sensitivity, and it can be applied for the discrimination of tumor cells from normal cells as well as the screening of anticancer drugs.

The principle of the telomerase assay is illustrated in

Scheme 1. We designed a specific primer with 18 nucleotides, and incubated the primers with cancer cell extracts in the presence of the nucleotides of dGTP, dTTP and Cy5-dATP at 37 1C for 1 h. The telomerase-induced extension reaction adds the repeat units of AGGGTT onto the 3 0-end of the primer and simultaneously assembles the Cy5 molecules into them, generating a Cy5-labeled primer. Subsequently, the Cy5-labeled primer can hybridize with the biotinylated capture probe to form a Cy5-labeled biotinylated double-stranded DNA (dsDNA).

Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced

Technology, Chinese Academy of Sciences, Shenzhen 518055, China.

E-mail: zhangcy@siat.ac.cn; Fax: +86-755-86392299; Tel: +86-755-86392211 † Electronic supplementary information (ESI) available: Details of experimental procedures and additional figures. See DOI: 10.1039/c5cc01600b

Received 23rd February 2015,

Accepted 12th March 2015

DOI: 10.1039/c5cc01600b www.rsc.org/chemcomm

ChemComm

COMMUNICATION

Pu bl ish ed o n 12

M ar ch 2 01 5.

D ow nl oa de d by

U ni ve rs ity o f M em ph is on 2 3/ 03 /2 01 5 03 :1 1: 01 .

View Article Online

View Journal

Chem. Commun. This journal is©The Royal Society of Chemistry 2015

Since the numbers of repeat units added at the ends of primer are usually 1, 2 and 3, which accounts for about 90% in all cases,17 we design the capture probe with 36 nucleotides which consist of two regions: one is the complementary region of primer, and the other is the complementary region of the added repeat units. The obtained Cy5-labeled biotinylated dsDNAs can attach onto the surface of streptavidin-coated QDs through the biotin–streptavidin interaction to form a Cy5-dsDNA–QD assembly (Scheme 1). Theoretically, the length between the adjacent bases in dsDNA is about 0.34 nm.18 When the numbers of repeat units added onto the ends of primer are 3, the distances between Cy5 molecules in the extended primer and the surface of streptavidin-coated QD is estimated to be 2.04 nm, 4.08 nm and 6.12 nm, respectively. Taking into account the radius of a streptavidin-coated QD (B7.5 nm),16a the corresponding QD-Cy5 separation distances are calculated to be 9.54 nm, 11.98 nm and 13.62 nm, within the efficient range of FRET (2R0 = 138.8 Å), 16a suggesting that efficient fluorescence resonance energy transfer (FRET) may occur between the QD donor and the Cy5 receptor.