An affinity capture involved enzymatic assay for thrombin by using peptide aptamers as affinity ligands on magnetic beads

Article abstract of DOI:10.1039/c3cc44400g

Here we present a sensitive and specific assay for thrombin through the affinity capture of thrombin with mRNA display generated peptide aptamers on magnetic beads and the subsequent thrombin-involved enzymatic reaction. Thrombin at 50 fM can be detected.

Full text of DOI:10.1039/c3cc44400g

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An affinity capture involved enzymatic assay for
thrombin by using peptide aptamers as affinity
ligands on magnetic beads†
Qiang Zhao*a and Jie Gaob
Cite this: Chem. Commun., 2013,
49, 7720
Received 11th June 2013,
Accepted 2nd July 2013
DOI: 10.1039/c3cc44400g
www.rsc.org/chemcomm
Here we present a sensitive and specific assay for thrombin through peptide is covalently connected with the expressed polypeptide
the affinity capture of thrombin with mRNA display generated peptide chain during the selection process.7 The mRNA display shows
aptamers on magnetic beads and the subsequent thrombin-involved advantages of a larger random sequence library and easy recovery
enzymatic reaction. Thrombin at 50 fM can be detected.
of the selected peptides.7
Thrombin is an important multifunctional enzyme involved
Affinity peptides (peptide aptamers) can be selected by in vitro in many physiological and pathological processes, such as blood
evolution from a combinatorial peptide library, specifically coagulation, thrombosis, inflammation, angiogenesis, tumor
binding with a target (e.g. small molecules or proteins).1,2 The growth and metastasis. Thrombin can be used as a therapeutic
binding affinity of peptide aptamers to proteins can be high, and a biomarker for diagnosis of some diseases, such as
and dissociation constants can reach low nanomolar or even pulmonary metastases and diseases associated with coagula-
picomolar levels.1,2 The peptide aptamers show great potential tion abnormalities.8 Numerous assays have been developed for
in biosensing and analytical applications,3 as alternatives to thrombin by using nucleic acid aptamers.4,9 The emergence of
antibody or nucleic acid aptamers.4 The peptide aptamers not peptide aptamers against thrombin provides another new
only show merits of easy generation and synthesis, small size, opportunity for the detection of thrombin. By using the mRNA
low cost, and being readily labelled with functional groups, as display technique, Raffler and coworkers successfully selected a
nucleic acid aptamers also show,4 but also possess the features thrombin-binding peptide aptamer (MGMGT CFFDR YDSAR
of proteins in rich interaction formats and diverse structures.1,2 DPGRL LSGSS MSGS) from an unbiased peptide library con-
For peptide aptamers, as many as twenty natural amino acids taining a random sequence of 15 amino acids.10 The generated
with a variety of properties can be incorporated into the peptide peptide aptamer does not show sequence similarity to the
sequence, significantly increasing the diversity of the inter- known natural thrombin-binding motif. This peptide aptamer
actions between targets and peptide ligands to enhance the exhibits a dissociation constant (Kd) of about 166 nM.10 This
success of the ligand selection for various targets.1,2,5 In con- peptide binds with a site close to the fibrinogen binding site of
trast, nucleic acid aptamers usually can only use four different thrombin.10 The binding of the peptide aptamer to thrombin
nucleotides as building blocks,4 causing less diversity in mole- does not interfere with the active site of thrombin. Since the
cular structure and interaction formats, and it may be difficult successful selection of the thrombin-binding peptide aptamer,
to select nucleic acid aptamers for some targets.2b,5 In addition, this peptide motif has not been applied to establish assays for
unnatural amino acids can also be introduced into the peptide thrombin.
sequence to further increase the diversity of functionalities.6
Herein, we present a simple, selective, and sensitive strategy
Many methods have been developed for the selection of for detection of thrombin by using the peptide aptamer against
peptide aptamers, including phage display, yeast two hybrid thrombin as affinity ligands on magnetic beads and the sub-
systems, ribosome display, mRNA display, in vitro compart- sequent enzymatic reaction of thrombin to explore the analytical
mentalization, etc.1,2,7 In mRNA display, mRNA of the expressed applications of the thrombin-binding peptide aptamer.11,12
Scheme 1 shows the proposed strategy for thrombin detection.
a Research Center for Environmental Science and Engineering, Shanxi University,
Taiyuan 030006, China. E-mail: chemzhaoq@hotmail.com; Fax: +86-351-7011011;
Tel: +86-351-7011011
Thrombin is first captured by the peptide aptamer conjugated on
magnetic beads. After washing and magnetic separation, the
magnetic beads are redispersed into the reaction solution that
contained the specific substrate of thrombin. Thrombin hydro-
lyzes the substrate to the product. The measurement of the
generated product allows the final detection of thrombin.
b College of Chemistry and Chemical Engineering, Shanxi University,
Taiyuan 030006, China
† Electronic supplementary information (ESI) available: Experimental details and
Fig. S1–S3. See DOI: 10.1039/c3cc44400g
c
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Scheme 1 Schematic diagram for detection of thrombin by using peptide aptamer
modified magnetic beads through affinity capture and subsequent enzymatic
reaction. Thrombin is selectively captured by the peptide aptamer on the magnetic
beads and separated from the sample mixture, and then thrombin catalyzes the
hydrolysis of the substrate to generate the product. The final measurement of the
product allows the sensitive and specific detection of thrombin.
Fig. 1 Detection of thrombin at varying concentrations by using the peptide
aptamer modified magnetic beads and fluorogenic substrate. (A) The recorded
fluorescene spectra of the generated products corresponding to varying concentra-
tions of thrombin ranging from 0 to 100 pM. The inset shows the obtained spectra
corresponding to thrombin at concentrations 0, 50 fM, 100 fM, 0.25 pM, 0.5 pM and
1 pM (from the bottom curve to the top curve). (B) The relationship between the net
increase of fluorescence intensity at 440 nm over the blank and the concentrations of
thrombin. 100 mL of sample was detected, and 2 h enzyme reaction was applied.
When a fluorogenic substrate of N-p-tosyl-Gly-Pro-Arg-7-amido-
4-methylcoumarin is used, thrombin converts the substrate to
the fluorescent product 7-amido-4-methylcoumarin, which
emits fluorescence at 440 nm with excitation at 370 nm.
We initially tested the influence of different binding solu-
tions on affinity capture in the assay, including the original
selection buffer solution, PBS buffer solution and Tris buffer
solution (Fig. S1A, ESI†). We chose PBS buffer solution as the
binding buffer solution in the assay. It is found that the
concentration of total NaCl higher than 200 mM is not favor-
able for the affinity binding between thrombin and the peptide
aptamer (Fig. S1B, ESI†). 30 min of incubation at room tem-
perature (20 1C) was sufficient for the affinity capture of
thrombin with the peptide aptamer coated magnetic beads.
Taking advantage of the signal amplification by enzymatic
reaction, the affinity preconcentration of targets by the peptide
aptamer on the magnetic beads, and the highly sensitive
fluorescence measurement, thrombin could be successfully
detected with high sensitivity by our demonstrated method.
When 2 h enzymatic reaction was applied, as low as 50 fM
thrombin in 100 mL could be detected (S/N > 3), corresponding
to 5 Â 10À18 mole of thrombin molecules. As shown in Fig. 1,
the linear dynamic range for thrombin detection was from
0.05 pM to 50 pM (log y = 1.032 + 1.053log x, R2 = 0.998). The
relative standard deviation (RSD) ranged from 3% to 10%. It is
expected that higher sensitivity can be further achieved when
longer-time enzymatic reaction is applied. For fast analysis,
a shorter time enzymatic reaction can be used to meet the
demand rapidly in spite of some sacrifice of sensitivity. Though
the peptide aptamer exhibited a Kd of about 166 nM as reported
in the literature,10 high local concentrations of the peptide
of the peptide aptamer, enabling the successful detection of
trace levels of thrombin. As a comparison, a similar sensitivity
was obtained for thrombin detection when 29-mer DNA aptamer
(Kd B 0.5 nM) modified magnetic beads were used instead,12–14
showing that the peptide aptamer is a good alternative to the
DNA aptamer. The obtained sensitivity of our assay for thrombin
is comparable to or better than that of assays reported using
nucleic acid aptamers.4,9,11–13
We assessed the specificity of the assay by detection of thrombin
along with other proteins and enzymes (Fig. 2). All the tested
proteins and enzymes did not interfere with the measurement
Fig. 2 Specificity test of the assay for thrombin by analyzing thrombin along
with other proteins including trypsin, proteinase K, chymotrypsin and elastase.
The concentration of thrombin was 25 pM. The concentrations of the tested
proteins were 50 pM. Thrombin (25 pM) in the presence of IgG (100 nM), HSA
aptamer on the magnetic beads enhanced the binding affinity (750 nM), or Hb (100 nM) was also analyzed.
c
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In summary, we described a simple, selective, sensitive assay
for thrombin by using the peptide aptamer as a new affinity
ligand. The assay combines the affinity capture of thrombin by
peptide aptamer modified magnetic beads and the subsequent
enzymatic reaction conducted by thrombin. Thrombin at 50 fM
could be successfully detected in assay buffer and the diluted
plasma sample. It is expected that the peptide aptamer can be
used for thrombin analysis with other detection strategies. The
use of a peptide aptamer provides new promise and opportunities
for assay development and shows great potential in biomarker
detection and biosensing.
Fig. 3 Detection of thrombin in the diluted human plasma sample. Blank (a),
20-fold diluted human plasma spiked with varying concentrations of thrombin:
0 pM (b), 0.2 pM (c), 2 pM (d), and 20 pM (e).
This work was supported by the grants from the National
Natural Science Foundation of China (Grant No. 21222503) and the
of thrombin. The presence of abundant proteins in blood, like Key Project of Chinese Ministry of Education (Grant No. 212020).
IgG (100 nM), hemoglobin (Hb, 100 nM), and HSA (750 nM),
Notes and references
did not affect the detection of thrombin at 25 pM. The good
selectivity results from the double selective procedure, the
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To examine the sequence specificity of the peptide, control
peptide modified magnetic beads were also tested. Thrombin
could not be well captured by the control peptide (MGMGT
CVRIA ASRIS TNNKY FSGSS MSGS), and very low signals over
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control peptide modified magnetic beads (Fig. S2, ESI†). Thus,
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human plasma as well as the binding buffer solution (Fig. 3).
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