Computational Lateral Flow Biosensor for Proteins and Small Molecules

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Computational lateral flow biosensor for proteins and small molecules: A new class of strip logic gates Junhua Chen, Zhiyuan Fang, Puchang Lie, and Lingwen Zeng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac301508b • Publication Date (Web): 05 Jul 2012 Downloaded from http://pubs.acs.org on July 10, 2012

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Analytical Chemistry

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Computational lateral flow biosensor for proteins and small molecules: A new class of strip logic gates

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Junhua Chen, Zhiyuan Fang, Puchang Lie, and Lingwen Zeng*

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Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and

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Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy

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of Sciences, Guangzhou 510530, China

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*Corresponding author:

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Fax: +86 20 32015245; Tel: +86 20 32015312;

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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Abstract

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The first example of strip logic gates (“OR” and “AND” functions) for proteins and

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small molecules has been constructed based on target-induced self-assembly of split aptamer

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fragments. Using thrombin and ATP as inputs, the corresponding split/integrated aptamers as

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molecular recognition elements, and gold nanoparticles as a tracer, the output signals can be

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directly visualized by observing the red bands on the test zones of the strips. The assay is

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simple, easy to perform, and cost-effective, allowing portable analysis at ambient temperature.

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The strip logic system is resistant to nonspecific interferants and can operate effectively even

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in human serum samples. Such logic strips hold great promise for application in intelligent

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point-of-care and in-field diagnostics.

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Electronic logic gates, transistor-based computational devices which perform binary

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arithmetic processing and Boolean logic circuits, form the basis of conventional computer

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microprocessors. By analogy, molecular logic gates can be viewed as molecular-scale

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computers that process chemical or physical “inputs” to generate “outputs” based on a set of

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molecular computation.1-3 Molecular logic designs aid chemical/biological sensing, small

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object recognition and intelligent diagnostics, which have attracted significant research

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interest.4-7 In order to identify ideal candidates which satisfy logic operations, extensive

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efforts have been dedicated to design diverse logic gates, such as OR, AND, NOR, NAND,

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XOR, INHIBIT, half-adder, and half-subtractor.8-21 Most of the molecular logic gates reported

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to date employ fluorescent, colorimetric, electrochemical, or electrochemiluminescent signals

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as

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instrument-dependent readout, and a lack of portability. In a step towards addressing these

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potential limitations, lateral flow strip biosensor (LFSB) is an economical and simple

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alternative approach, which has recently attracted considerable attention.22-28 The

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LFSB-based methods possess several benefits including a user-friendly format, long-term

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stability, short assay time, and cost-effectiveness. Moreover, these strip biosensors eliminate

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complex analysis procedures that involve expensive instrumentation and minimize the

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requirements for highly qualified personnel. The semi-quantitative assay is easy to perform

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by visually observing the color intensity of the test zone, and the quantitative data can be

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obtained by recording the optical responses with a portable “strip reader”. Recently, our

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group has successfully developed LFSB for the detection of nucleic acids,29-31 proteins,32

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cancer cells,33 and heavy metal ions.34 To explore new dimensions in molecular logic gates, it

their

outputs,

which

often

suffer

from

cumbersome

3


handling

procedures,


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would be of great interest to combine strips with logic gates. Such combination is appealing

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in the fabrication of intelligent point-of-care devices and the design of molecular logic

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systems for on-site applications.

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Aptamers are artificial functional oligonucleotides that have high affinity and specificity

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toward their targets. It has been demonstrated that some aptamers can be split into two

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fragments without significant perturbation of their ligand-binding abilities.35-37 In the current

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work, we split the 15-mer anti-thrombin aptamer and 27-mer anti-ATP aptamer into two

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fragments (subunits), respectively, according to the findings of Chen’s and Plaxco’s

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groups.14,38,39 One subunit of thrombin aptamer was coupled with one subunit of ATP aptamer,

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and the other subunit of thrombin aptamer was combined with the other subunit of ATP

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aptamer, which obtained two integrated oligonucleotides. In the absence of targets, the

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integrated strands do not interact with each other. In the presence of respective or both targets,

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however, a tricomponent supramolecular aptamer complex is generated based on

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target-induced self-assembly of split aptamer fragments. By the appropriate labeling of the

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aptamer subunit with gold nanoparticles (AuNPs), aptamer subunits-target binding events can

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be directly visualized with the naked eye using the strip sensing platform. The molecular

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recognition elements of our constructed strip logic gates are split aptamers, which are

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different

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aptamer-crosslinked hydrogels for logic gates operation visualized with AuNPs.21 However,

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the detection mechanism is based on target-induced conformational changes of intact

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aptamers. Aptamer-based protein detection visualized with AuNPs in a lateral flow strip was

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reported by Xu et al.28 However, the sandwich configuration requires special target molecule

from previously

published

work.

For

example,

4


Yin

et

al.

reported

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which has two binding sites to interact with two intact aptamers simultaneously. So, our work

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is distinctive and a new exploration of aptamer-based logic gates. Herein, for the first time,

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we fabricate a strip logic system (“OR” and “AND” logic gates) for proteins and small

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molecules based on target-induced linkage of split aptamer fragments. Our work not only

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provides a “smart” and flexible logic platform for thrombin and ATP sensing, but also can

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expand the application for other ligand assays, such as adenosine monophosphate (AMP),

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theophylline, and cocaine.40-42 It was reported that the aptamers of these ligands can also be

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split in halves that can self-assemble into a supramolecular aptamer fragments/target complex

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in the presence of the target.

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As a first test we have designed a two-analyte “OR” logic gate (Scheme 1). The two

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integrated DNA are defined as OR1 and OR2, respectively (the split thrombin aptamer

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fragments are marked in blue; the split ATP aptamer fragments are marked in red). The two

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inputs are thrombin and ATP, with the absence and presence of each molecule defined as “0”

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and “1”, respectively. The output is the color of the test zone, with the absence and presence

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of a red band on the test zone defined as “0” and “1”, respectively. OR1-conjugated gold

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nanoparticles (OR1-AuNPs) are used as detection probes and biotin modified OR2

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(OR2-Biotin) are used as capture probes. Relatively small AuNPs (~15 nm diameter) were

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used in the current work because they are stable and can increase the sensitivity and

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reproducibility of strip biosensors.29 The LFSB consists of three components: sample pad,

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nitrocellulose membrane, and absorption pad. Conjugate pad was not used for the storage of

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DNA-AuNPs conjugates. If conjugate pad is used, the incubation time ( 3 min) wouldn’t be

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enough for the target-induced self-assembly of the split aptamers and the targets, which

5



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would then lower the detection sensitivity. As an alternative, we chose to store the

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DNA-AuNPs conjugates in centrifuge tubes at 4

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streptavidin-biotin-control DNA (partially complementary to the OR1) were dispensed on the

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nitrocellulose membrane to form the test and control zone, respectively. In the (0,0) state of

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the “OR” gate, the OR1 and OR2 exist predominantly in the dissociated form. The sample

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solution containing OR1-AuNPs and OR2-Biotin is applied on the sample pad. The solution

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migrates along the strip by capillary action and the OR2-Biotin are captured on the test zone

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via the streptavidin-biotin interaction, while the OR1-AuNPs are captured on the control zone

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via the hybridization between the OR1 and the pre-immobilized streptavidin-biotin-control

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DNA. The accumulation of AuNPs on the control zone is visualized as a characteristic red

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band, indicating that the LFSB is working properly. In the absence of any inputs (0,0), no

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OR1-AuNPs is captured on the test zone and no red band is observed on the test zone,

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indicating that the output is 0. In the presence of either (1,0; 0,1) or both inputs (1,1), the

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OR1-AuNPs would combine with OR2-Biotin to form G-quadruples structure at either or both

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sides of the integrated DNA strands to bind thrombin and/or ATP. Such target-induced

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conjunction of split aptamer fragments, in turn, allows the detection probes OR1-AuNPs to be

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captured on the test zone through the OR2-Biotin-streptavidin interaction. The accumulation

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of AuNPs on the test zone is visualized as a characteristic red band, indicating that the output

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is 1. The excess OR1-AuNPs would be captured on the control zone by the hybridization

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between OR1 and the control DNA probe, thus forming a second red band. Therefore, either

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or both inputs can induce a red band on the test zone and obtain a true value of output, giving

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rise to an “OR” logic gate.

o

C before use. Streptavidin and

6


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2 3

Scheme 1. Schematic illustration of the design strategy of the strip “OR” logic gate using

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thrombin and ATP as inputs. SA, streptavidin; CZ, the control zone; TZ, the test zone.

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Figure 1A presents typical photo images of the “OR” gate, and Figure 1B and 1C show

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the corresponding calculation results. Much larger peak areas were obtained when the logic

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circuit was subjected to either or both inputs than that the original state (0,0). A truth table is

4

given in Figure 1D.

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Figure 1. (A) Photograph of the LFSB in “OR” gate. (B) Their corresponding optical

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response features. (C) Peak areas of the red bands on the TZ. (D) A truth table of the “OR”

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logic gate. The concentrations of thrombin and ATP are 2 M and 2 mM (Figure S1 and S2,

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Supporting Information), respectively.

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Encouraged by these results, we sought to create an “AND” logic gate using similar

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design strategies (Scheme 2). Biotinylated AND1a (AND1a-Biotin) and AuNPs modified

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AND1b (AND1b-AuNPs) were used as capture probes and detection probes, respectively.

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Streptavidin and streptavidin-biotin-control DNA (partially complementary to the AND1b)

5

were dispensed on the nitrocellulose membrane to form the test and control zone, respectively.

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Only the presence of both inputs (1,1) could cause the capture probes (AND1a-Biotin) to

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combine with the detection probes (AND1b-AuNPs) through AND2 based on target-induced

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conjunction

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(AND1a-Biotin-AND2-AND1b-AuNPs) are captured on the test zone through the reaction

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between the biotin and the pre-immobilized streptavidin. The excess of AND1b-AuNPs are

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captured on the control zone by the hybridization between the AND1b and the

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streptavidin-biotin-control DNA. Thus, red bands are formed on the test zone and control

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zone due to the accumulation of AuNPs. Accordingly, the output reads 1. In the absence of

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any inputs (0,0) or in the presence of either input (1,0; 0,1), the AND1a-Biotin and the

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AND1b-AuNPs exist mainly in the dissociated state and can hardly interact with each other.

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No red bands would be observed on the test zone in the pattern of such input combinations

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due to the failure of capturing the AND1b-AuNPs on the test zone and the output reads 0. A

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red band is always formed on the control zone of the strip to confirm the proper functioning

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of the test. The system performs the “AND” logic circuit, and the output signal (a red band on

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the test zone) is generated (true value “1”) only if the system is subjected to the both inputs

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simultaneously.

of

split

aptamer

fragments.

The

22

9


formed

complexes


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1

2 3

Scheme 2. Schematic illustration of the design strategy of the strip “AND” logic gate using

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thrombin and ATP as inputs. SA, streptavidin; CZ, the control zone; TZ, the test zone.

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1

Figure 2A presents typical photo images of the “AND” gate, and Figure 2B and 2C show

2

the corresponding calculation results. Much larger peak areas were obtained when the logic

3

circuit was subjected to both inputs than other input combinations. A truth table is given in

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Figure 2D.

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Figure 2. (A) Photograph of the LFSB in “AND” gate. (B) Their corresponding optical

7

response features. (C) Peak areas of the red bands on the TZ. (D) A truth table of the “AND”

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logic gate. The concentrations of thrombin and ATP are 2 M and 2 mM, respectively.

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To validate the specificity of the “OR” and “AND” logic gates, non-target molecules,

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such as cytidine triphosphate (CTP), uridine triphosphate (UTP), hemoglobin, and lysozyme

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were used as negative control inputs. As shown in Figure S3 (Supporting Information), the

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“OR” logic gate obtained a red band on the test zone (output = 1) using thrombin or ATP as

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stimulus input but no red band was visualized on the test zone (output = 0) using any of the

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control inputs. As shown in Figure S4 (Supporting Information), the “AND” logic gate

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yielded a red band on the test zone (output = 1) in the presence of both thrombin and ATP

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inputs but no red band was observed on the test zone (output = 0) in the presence of other

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input combinations. These results demonstrate that the logic gates based on target-induced

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conjunction of split aptamer fragments are resistant to nonspecific interferants.

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The practical applicability of the proposed logic system was investigated by operating

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the “OR” and “AND” logic gates in human serum samples. Serum samples were prepared by

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diluting to 10% in dilution buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, pH 7.5).

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Thrombin and ATP spiked in 10% human serum were used as inputs to perform the logic

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operation for real biological samples. Sample solution containing the targets, the detection

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probes, and the capture probes was incubated for 30 min at room temperature and then

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applied to the sample pad of the LFSB for logic analysis. As shown in Figure S5 (Supporting

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Information), without the spiked thrombin and ATP (0,0), no red band is observed on the test

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zone in the “OR” gate (output = 0). While serum samples spiked with either thrombin (1,0) or

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ATP (0,1), or both the inputs (1,1), a red band can be visualized by the naked eye on the test

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zone (output = 1). As shown in Figure S6 (Supporting Information), a red band is formed on

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the test zone only if the “AND” gate is subjected to the two spiked inputs together (1,1). In

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the absence of both inputs (0,0) or in the presence of either input (1,0; 0,1), the system fails to

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produce a red band on the test zone. It indicates that the strip logic system performs well even

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in relatively complex sample matrices and is not affected when the inputs existed in clinical

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samples. The results also definitely illuminate the potential application of the logic design in

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clinical diagnostics.

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In summary, we have introduced, for the first time, a strip logic system (“OR” and

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“AND” logic gates) for proteins and small molecules based on target-induced self-assembly

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of split aptamer fragments. Using the split/integrated aptamers as molecular recognition

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elements, thrombin and ATP as inputs, and gold nanoparticles as a tracer, the outputs can be

10

directly visualized by observing the red bands on the test zone of the strips. Such logic gate

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system possesses several distinctive advantages that are listed below. (1) The assay is simple,

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easy to perform, cost-effective and does not demand either technical expertise or expensive

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sophisticated instruments. The logic operation with a user-friendly format is readily

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performed in local clinical laboratories. (2) The strip outputs can be unambiguously

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determined by the presence or the absence of visible red bands on the test zone, and each

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measurement can be readily finished within 10 min. This strip logic system can be developed

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into rapid test kits, which allows portable analysis at ambient temperature. (3) Colored

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samples can also be used as inputs in the strip logic operations, and do not affect the

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detectability and performance of the biosensor. While such colored inputs are questionable

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for use in colorimetric logic gates. (4) The logic strips also exhibit excellent selectivity and

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are capable of monitoring the targets in human serum solutions, demonstrating the robustness

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of the logic system. In view of these advantages, our work not only introduces a new concept

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for devising strip logic gates, but also provides a “smart” and flexible logic sensing platform

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for point-of-care and in-field diagnostics.

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Acknowledgments Financial support was provided by the Ministry of Science and Technology (No. 2008ZX10004-004, No. KSCX2-YW-R-164, and No. 2009ZX1004-109).

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Supporting Information Available Experimental details, sensitivity analysis, and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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1

For TOC only

2

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Scheme 1. Schematic illustration of the design strategy of the strip “OR” logic gate using thrombin and ATP as inputs. SA, streptavidin; CZ, the control zone; TZ, the test zone. 296x394mm (300 x 300 DPI)



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Figure 1. (A) Photograph of the LFSB in “OR” gate. (B) Their corresponding optical responses features. (C) Peak areas of the red bands on the TZ. (D) A truth table of the “OR” logic gate. The concentrations of thrombin and ATP are 2 uM and 2 mM (Figure S1 and S2, Supporting Information), respectively. 234x185mm (300 x 300 DPI)


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Scheme 2. Schematic illustration of the design strategy of the strip “AND” logic gate using thrombin and ATP as inputs. SA, streptavidin; CZ, the control zone; TZ, the test zone. 296x415mm (300 x 300 DPI)



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Figure 2. (A) Photograph of the LFSB in “AND” gate. (B) Their corresponding optical responses features. (C) Peak areas of the red bands on the TZ. (D) A truth table of the “AND” logic gate. The concentrations of thrombin and ATP are 2 uM and 2 mM, respectively. 241x197mm (300 x 300 DPI)


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Computational Lateral Flow Biosensor for Proteins and Small Molecules

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