分子钳子：基于抗体的均相蛋白传感器
美国分析化学杂志刊登了一篇文章：Molecular Pincers: Antibody-Based Homogeneous k-j SS:b b+S
Protein Sensors。
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摘要：)jI a I0Y v^8X\@
[quote]
protein sensor design (molecular pincers) that allows
rapid and sensitive detection of a specific protein in
solution. In the presence of the target protein theseN:@ F.M&ri4H p
sensors produce fluorescence signal derived from targetdependent
annealing of short complementary fluorochrome-
labeled oligonucleotides attached to a pair of
target-specific antibodies via nanometer-scale flexible!s._D S:g G Q)\*E
linkers. The sensors allow near-instantaneous detection
of the target with sensitivity and specificity approaching
that of enzyme-linked immunosorbent assay (ELISA) but/i D)Q `'Y2a
requiring no sample manipulation other then the addition
of the sample to the sensor mix. We used cardiac troponinDqX7j@*z_l&h2h6V
I and C-reactive protein as the targets to validate theseB H6` ]$O%[
desirable properties of the sensors. Due to the availability
of antibodies to thousands of interesting targets and the!V&E kT:@\'K
straightforward design blueprint of the sensors we expectU.Xc0p$g6KNu A
their wide-ranging applications in research and medical b"S A q"t%["L5V _ n)B
diagnosis, especially when simplicity, high throughput, U\ APV:x
and short detection time are essential.
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前言：&F2y!@ L*`
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Detection and determination of a level of a specific protein is { ]`%X(F$?'p J3u t\
the most commonly performed assay in biomedical research and
clinical diagnosis. A number of powerful techniques utilizing
antibodies have been developed for detecting proteins. ELISAS] L7jq p6uw"F*?9A
(enzyme-linked immunosorbent assay) represents the gold standard
of specificity and sensitivity of protein detection.1–5 The
outstanding utility of antibody-based assays was a motivation for%l8`o]2a8m2F
the development of thousands of antibodies. Despite the unquestionable6`)r!g P)O2J
value of currently available immunoassays, there is an{RB1[j.e?
significant demand for further development of antibody-basedM}%X Z+rv"w*x
protein detection methodologies with a specific emphasis on
increasing the speed of the detection, improving sensitivity,&z:S0Bv I
increasing multiplexing capabilities, and reducing the cost of the J af5{5K(i @
assays. Reducing the complexity of assays such that they could
be adaptable to point-of-care use is also a worthy goal. The currentamf@ \9~
most widely used specific protein detection methodology, ELISA,*K KF"At5A
is a heterogeneous assay requiring a number of manipulations
(coating of the plates with antibodies, incubation with a sample,
extensive washings, adding reagents for enzyme amplification
reaction, and reading of the signal) that require up to several hours i"F P` gS%\4Y
to perform. The goal of the work presented here was to develop$D)c4km(Po"s2v `
homogeneous antibody-based protein sensors that would retain
most of the positive aspects of ELISA but would eliminate the
complexity and greatly reduce the time necessary for protein
detection.!Dj8CH O L1G
Design of these sensors (Figure 1A) utilizes target proteininduced
increase of local concentration of complementary oligonucleotides_j$lD+y'u#g2vn-x2{K
with which the pair of the antibodies recognizing
nonoverlapping epitopes of the protein are labeled. This increase"h!Ei9k;\ x-L R
in the local concentration results in the annealing of the complementary+f)\ f:b2XL v3Eo_] M
oligonucleotides producing efficient fluorescence resonance
energy transfer (FRET) between the fluorescence probesv3im(|/psn C3T
incorporated into the oligonucleotides. This FRET signal can be a+j%nGtuO(~
used for sensitive, near-instantaneous detection of the target
protein. Oligonucleotide-labeled antibodies have been previously @ ?#n/M0j'K i(`z y
employed in conjunction with polymerase chain reaction (PCR)"w/Fg.]m$g:F Q |Rt
or rolling circle amplification to achieve great enhancements in
sensitivity of immunoassays.6–10 The emphasis of our sensor
design was on homogeneous and rapid detection.
Since we expected the speed and reduced complexity to be
the major attributes of our new protein sensors, we have chosen
cardiac troponin I as an example protein target for sensor C"? vD(X1N
development. Cardiac troponin is a perfect example of a target-N\ g-T$h\-T
protein requiring a rapid, specific, and inexpensive assay compatible-M ?2Q#L f'v5G,e {$S
with bedside applications. Troponin is the contractile regulatingkK7X*s#q k+_"o
protein complex of striated muscle.11 Cardiac troponin I test
has been extremely useful in the differential diagnosis of patients
admitted to the emergency room with chest pains.12–14 The levels%d6}+aP:AB/g*F7h
of troponin I are very low in normal serum. After acute myocardial'[V]W.j
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