Biochips – fundamentals and applications - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 319

(a) (b) (c)

(d)

FIGURE 11.11 Schematics of the RCA processes with an arrowhead symbolizing the DNA polymerase.
(a) Traditional phenomenological view of RCA going on a free DNA minicircle with the use of a single
primer. If the DNA target is used to prime the RCA reaction, amplifi cation products are fi xedly linked to tar-
get molecules. For surface-attached targets, these products will be immobilized on solid-phase. (b) More real-
istic representation of the RCA-generating complex that resembles here a bow with a string. (c) RCA-based
diagnostics of probe amplifi cation with a target-unrelated primer. In some cases, the pseudo- or true topo-
logical linkages between a DNA minicircle and the marker/target DNA site may affect the rolling replication.
A circular probe should then be released from the DNA target following the hybridization. (d) Simplifi ed
(not in scale) representation of initial stages of the double-primed RCA (for more detailed schematics see).
In these reactions, the second primer is used, which is complementary to the original RCA product; DNA
polymerases capable of strand-displacement synthesis are also necessary.

Tyramide signal amplifi cation (TSA)
TSA is a technique to increase the S/N ratio by using an enzyme-mediated detection
method that utilizes the catalytic activity of horseradish peroxidase (HRP) to acti-
vate and deposit a reactive tyramide-labeled tag. Alfonta et al. [23] labeled liposomes
with biotin and HRP. The liposomes are then used as a probe to amplify the sensing
of antigen-antibody interactions or oligonucleotide-DNA binding. According to Am
[24], this indirect labeling method increases the strength of the signal amplifi cation by
ten- to 100-fold as compared to the conventional avidin-biotinylated enzyme complex
(ABC) procedures, thus allowing very low levels of nucleic acid to be discerned. The
initial stage of TSA is shown in Fig. 11.12; the conventional ABC method, together
with pretreatment procedures and optimal antigen retrieval, is used. Next, the reac-
tive biotinylated tyramide is formed through the catalytic reaction HRP and hydrogen
peroxide catalyses in Fig. 11.13. Covalent coupling of the produced, highly reactive,
short-lived tyramide radicals to the electron-rich moiety of protein tyrosine residues
in the vicinity of the HRP reaction site results in minimal diffusion-related loss of sig-
nal localization. The fl uorescent signal can be immediately detected, resulting in both
excellent spatial resolution and high signal intensity.

11.2.5 Detection and data analysis
11.2.5.1 Detection technologies
Fluorescent detection technology applicable to biochips is evolving rapidly, resulting in
detection instruments that are more powerful, user-friendly, and less expensive. Most
systems employ photomultiplier tube (PMT) technology in conjunction with multiple
colors, lasers, and a variety of fi lters. It is essentially a fl uorescent microscope that