James P. Chambers1,*, Bernard P. Arulanandam1, Leann L. Matta2, Alex Weis3, and James J. Valdes4




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Antibody based recognition

With the notable exception of the glucose sensor, the

majority of rapid detection systems employ antibodies

for recognition, identification and quantitation of target

analytes. Antibodies have been used extensively for

detection purposes; however, their popularity increased

significantly following Kohler and Milstein’s seminal work

establishing monoclonal antibody (MoAb) technology

(Jayasena, 1999). Using cell clones that specifically

produce MoAbs of choice, large quantities of antibody

can now be produced. Antibody recognition elements

make use of the sensitivity and specificity of bimolecular

antibody–antigen interactions. The major advantage

of antibody sensor biorecognition elements is that the

immunogen, i.e. target, need not be purified prior to

detection. A variety of signal transduction (optical and

electrochemical) techniques have been developed and

the most useful has been enzyme-fluorescence based

with catalytic turnover resulting in amplification of signal,

thus increasing the sensitivity of the assay.






4 Chambers et al.


Like many routinely used diagnostic assays, the

majority of current PSA (prostate-specific antigen)

assays are variations of enzyme-linked immunosorbent

assays (ELISA) reporting via the specific formation

of PSA immune complex. Wu and coworkers’ (2001)

observation of nanomechanical motion generated by

protein ligand interactions on microcantilevers has led to

immobilization of PSA antibody recognition element for

detection of PSA in serum (Fig. 4). Molecules adsorbed

on a microcantilever cause vibrational frequency changes

referred to as “curling” due to adsorption stress on one

side of the cantilever. Surface plasmon resonance based

optical transduction by noble metals has also been used

as basis of antibody recognition element assays (Hirsch

et al. 200 ). Such technology readily lends itself to well-

established array microfabrication techniques, thereby

offering the promising prospect of high throughput,

biosensor based analysis of clinical samples.

Recombinant antibodies consisting of genetically

manipulated fused antigen binding domains (Fab fragment)

of common antibodies are now available (Emanuel et

al., 2000). When compared to polyclonal or monoclonal

antibodies, generation (expression and purification)

of recombinant antibodies is less expensive and time

consuming. A number of recombinant antibodies have

been shown to be useful for detection and identification

of HIV, Hepatitis B and C, Simian immunodeficiency,


to that of high-sensitivity immunodetection of proteins

was achieved by establishment of immuno-PCR (Sano et

al., 1992) resulting in detection of clinically relevant tumor

markers, viral proteins, pathogens (microorganisms),

toxins and metabolites (Niemeyer et al., 2005). Immuno-

PCR takes advantage of specific, conjugated recognition

elements comprised of an antibody and DNA marker

fragment, combining the versatility of enzyme-linked

immunosorbent assays (ELISAs) with the amplification

power and sensitivity of the PCR (Fig. 5, Frame A). As a

consequence, the limit of detection of a given ELISA is in

general enhanced 100–10 000 fold by the use of PCR as

a signal amplification system (Fig. 5, Frame B). Although

Immuno-PCR is now a well-established technique for

routine applications in both fundamental and clinical

applied immunological research, to date the PCR step

must be carried out using a PCR machine. However,

miniaturized PCR devices using microfluidic channels

and reaction chambers are now described in the literature

(Koh et al., 200 ; Gulliksen et al., 2004) making possible

development of a “chip based” immuno-PCR biosensor.

Additionally, DNA–DNA hybridization detection has been

reported using cantilever based recognition elements

which could be applicable to Immuno-PCR (Hansen et

al., 2001; Wu et al., 2001).

Antibodies are currently being used in nontraditional

immunoreagent mediated sensing schemes. One such

Ebola, Rabies, Epstein–Barr, and Measles viruses as

well as biological agents such as botulinum neurotoxin

A/B (O’Brien et al., 2001; Petrenko and Sorokulova,

2004). Benhar and coworkers have demonstrated the

usefulness of carbon electrode immobilized single chain

(scFvs) recombinant antibodies specific for Listeria

monocytogenes, and the MtKatG enzyme expressed

by Mycobacterium tuberculosis in an electrochemical

biosensor (Benhar et al., 2001).

A clever marriage of the well-established polymerase

chain reaction (PCR) nucleic acid detection methodology


Fig. 5 General setup of immuno-PCR (IPCR) is similar to that of antigen

(Ag) detection using ELISA. Frame A, Comparison of ELISA and IPCR.

Fig. 4 Diagram of interactions between target and probe molecules on a

microcantilever beam.

Frame B, Typical comparison of dose-response curves for ELISA (closed

circles) and analogous IPCR (open circles).









Biosensor Recognition Elements 5


technology is that of the “ion channel switch” which

involves a self-assembling synthetic biomembrane.

This biosensing element is a two molecule layer, self-

assembled membrane structure modeled after the ion-

channel peptide gramicidin. The binding of target analyte

to antibody tethered to the channel alters the population

of conduction ion channel pairs within the membrane,

resulting in a change in membrane conduction and an “on/

off” response resulting from channel closure or distortion

(Krishna et al., 200 ).

Another non-traditional but novel immunoreagent

sensing scheme is that of antibody dendrimers (Yamaguchi

and Harada, 200 ). As shown in Fig. 6, the antibody

dendrimer complex consists of an immunoglobulin core

(IgM) and chemically modified IgG branches. Addition

of antibody to the divalent antigen–antibody complex

immobilized on the sensor surface results in increased

SPR signal intensity.


Aptamer based recognition

Biosensors using aptamers as biorecognition elements

are referred to as aptasensors. Aptamers are nucleic

acid ligands (RNA, ssDNA, modified ssDNA, or modified

RNA) that are isolated from libraries of oligonucleotides

by an in vitro selection process called SELEX (Systematic

Evolution of Ligands by EXponential enrichment)

(Ellington and Szostak, 1990; Tuerk and Gold, 1990).

These DNA/RNA ligands are thought to recognize their

target primarily by shape (i.e. conformation) and not

sequence (Lim et al., 2005). Since they are short, single-

stranded oligonucleotides, they are capable of folding into

three-dimensional structures due to their self-annealing

properties. Aptamers bind with high affinity and specificity

to a broad range of target molecules, and have proven

suitable for analytic and diagnostic applications (Luzi

et al., 200 ). Since aptamers are synthetically evolved

in the SELEX process, their potential range of use is

virtually unlimited. As shown in Fig. 7, a library of DNA

oligonucleotides containing a portion of randomized

sequence is synthesized. The library is then converted

into dsDNA by PCR and into RNA by in vitro transcription

using the T7 RNA polymerase. After incubation of the

target analyte with the nucleic acid pool, the non-specific

or low-affinity binding nucleic acid molecules are removed


Fig. 6 A proposed structure of an antibody dendrimer complex.

Fig. 7 Scheme of in vitro selection of an RNA aptamer (SELEX).


by washing steps and the captured RNA molecules

are eluted, recovered and amplified by RT-PCR and

subsequently transcribed back to RNA. The whole cycle

is repeated until obtaining a specific population of high

affinity binding RNA which is isolated and characterized.

Aptamers have been produced against a wide range

of targets including small molecules, proteins and whole

cells, making them very useful for detection purposes.

These nucleic acid recognition elements are more

flexible than their protein counterparts. Predominantly

unstructured in solution, aptamers fold upon associating

with their ligands into molecular architectures in which

the ligand becomes an intrinsic part of the nucleic

acid structure. Due to their high binding affinity, simple

synthesis, easy storage, and wide applicability, aptamer

sensor recognition elements are emerging as a new class

of molecules that rival commonly used antibody biosensor

recognition elements.

The efficacy of aptamers has been shown on a

number of biosensing platforms. Stadtherr and coworkers

have demonstrated the feasibility of a DNA aptamer-

based biochip for protein detection (IgE and specific

anti-IgE antibodies) (Stadtherr et al., 2005). Cantilever

surfaces have been functionalized with aptamers

(Savran et al., 2004). Liss et al. (2002) reported label-

free detection of IgE using aptamers and a quartz crystal

microbalance system. Detection of HIV-1 Tat protein

(trans activator of transcription) has been achieved using

RNA aptamer recognition element based quartz crystal

biosensor (Minunni et al., 2004). Recently, Gronewold

and coworkers (2005) devised an anti-thrombin aptamer

surface acoustic wave (SAW) biosensor for monitoring

blood-coagulation cascade complex formation.

Catalytic aptamers called aptazymes are relatively

new to the recognition element theme and may prove

especially useful for capturing and thus monitoring key









6 Chambers et al.


metabolic intermediates of diagnostic value whose

concentration might be very low (Hesselberth et al.,

200 ). Aptazymes (RNAzymes and DNAzymes) are

aptamers possessing allosteric properties that transduce

recognition of target analytes into catalytically generated

observable signals. With the development of functionalized

gold nanoparticles, DNAzymes have become especially

attractive biosensor recognition elements because many

analyte-dependent DNAzymes have been isolated (Table

1). DNAzymes may be very useful in monitoring diseases

such as diabetes and Alzheimer’s as well as chemical

warfare agents due to pathopneumonic changes in metal

ion concentrations. Also noteworthy, DNAzymes, like

RNAzymes, can be repeatedly denatured without losing

catalytic/binding abilities. Furthermore, DNA is relatively

inexpensive to produce and can be easily derivatized.

Aptazymes are easily engineered and can detect diverse

classes of biologically relevant molecules from small

organics to proteins, and their high signal-to-noise

ratios make them ideal for array formats, which have

been shown to apprehend and quantitate a wider range

of analyte classes than would be possible with typical

antibody-based sandwich assays (ELISA arrays) (Kirby

et al., 2004).

A key development in aptamer-based sensors is

that of “signaling or ‘molecular beacon’ aptamers,” which

have the ability to report directly following binding to

their specific targets. Molecular beacons have become

a class of DNA/RNA probes used in chemistry, biology,

biotechnology and medical sciences for molecular

recognition. However, this theme has now been adapted

to a variety of nucleic acid formats including aptamers

and nucleic acid peptides. These beacons act like

switches that are normally closed or “off.” Binding induces

conformational changes that open the hairpin and, as a

result, fluorescence is turned “on.” The stem structure

holds the fluorophore and the quencher in close proximity

to one another, preventing the fluorophore from emitting

a signal as a result of resonance energy transfer (Tyagi


and Kramer, 1996). Once the single stranded loop portion

hybridizes to the target, the stem melts and the resulting

spatial separation of the fluorophore from the quencher

leads to an enhancement of fluorescence signal (Fig.

8). Molecular beacon aptamers have been made

against clinically relevant molecules such as thrombin

(Nutiu and Li, 2004). Of clinical relevance in diagnostic

screening is the fact that it has been estimated that 60%

of all humans will have been affected by gene mutations

in their lifetime and biorecognition elements capable

of differentiating between two target DNA sequences

differing by only a single nucleotide (Wang et al., 2002)

will contribute significantly to high-throughput mutation

detection. Additionally, signaling aptamers have the ability

to indicate the presence of non-nucleic acid analytes such

as proteins and small organic compounds (Rajendran and

Ellington, 2002) which make them very useful for general

detection purposes.

Because the precise target binding sites and the

conformational changes of the aptamers are generally

unknown beforehand, it is not easy to design donor/

acceptor labeling strategies. Therefore, efforts have been

made to develop alternative methods without aptamer

labeling. Stojanovic and Landry (2002) developed a

calorimetric aptamer sensor for cocaine using specific

dyes which, when displaced by the analyte cocaine,

result in attenuation of absorbance proportional to the

concentration of cocaine added. Jiang and coworkers

have successfully used 1 RNA and 2 DNA aptamers

specific for three disease-related proteins of diagnostic

importance, i.e. IgE, PDGF-BB and (Jiang

et al., 2004). The method is simple, with no need for

aptamer recognition element immobilization, and takes

advantage of a sensitive luminescence change upon

aptamer-protein binding via a ruthenium phenazine

molecular light switch. The Ru-(phen)2(dppz)2+ “light

switch” complex does not luminesce in aqueous solution.

However, when bound to dsDNA, the interaction protects

the phenazine nitrogen from water, leading to intense

emission. Thus, the folded structures of the aptamers


Table 1 Various DNAzymes

Reaction

RNA transesterification


DNA cleavage

DNA ligation

RNA ligation

DNA phosphorylation

5′,5′-pyrophosphate formation

Porphyrin metallation


Cofactor

Pb2+

Mg2+

Ca

Mg

None

L-Histidine

Zn

Cu

Cu2+ or Zn2+

Mn

Ca

Cu2+

None


kmax(min-1)a


0.01

0.08

10

0.01

0.2

~40

0.2

0.07

2.2

0.01

0.5

1.


kcat/kuncat

105

105

105

>105

108

106

>105

>106

105

>106

109

>1010

103

allow intercalation of [Ru(phen)2(dppz)]2+ resulting in

luminescence which is blocked by aptamer binding to

target protein.

The primary limitation of the use of aptamers,

specifically RNA aptamers, as recognition elements is

their sensitivity to pyrimidine specific nucleases that are

abundant in biological fluids. However, specific chemical

modification of the ribose ring at 2′-position (Pieken

et al., 1991) of pyrimidine nucleotides (2′-amino and 2′

fluoro functional groups) results in significant stability and

protection. Additionally, 2′-amino and 2′-fluoro CTP and

UTP can be incorporated into in vitro transcribed RNA,

thus modifications can be introduced directly into the

combinatorial library.

The advantages of aptamer recognition elements

also make them very attractive for biosensor array

formats. Lee and Walt (2000) have adapted aptamers to

high-density fiber-optic arrays. McCauley and coworkers

have generated a small aptamer sensing array that relies

on scanning fluorescence anisotropy measurements for

a Reactions catalyzed by DNAzymes that were isolated from in vitro ex-

periments. kcat/kuncat is the rate enhancement over uncatalyzed reaction.

detection of several proteins with relevance to cancer

(inosine monophosphate dehydrogenase, IMPDH;
1

2+

2+

2+

2+

2+

2+




Biosensor Recognition Elements 7


Fig. 8 Structural characteristics of molecular beacon probes. At the far left is shown a typical molecular beacon DNA probe. Shown to the right of the typical

molecular beacon DNA probe is the molecular beacon working principle. The two spheres on the molecular beacon (center) represent quenched Tamara

and Dabcyl moieties in contrast to unquenched, i.e. fluorescing (right).



vascular endothelial factor, VEGF and basic fibroblast

growth factor, bFGF) (McCauley et al., 200 ). Kirby and

coworkers (2004) have integrated aptamer arrays with

a device that could also deliver samples and perform

complex assay procedures applicable to clinical sample

sensing.

1   2   3   4

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