WO2017223313A1

WO2017223313A1 - Aptamer-based compositions and methods for extraction and detection of microbial contamination

Info

Publication number: WO2017223313A1
Application number: PCT/US2017/038745
Authority: WO
Inventors: Ehsan SALAMIFAR; Baichen LI; Abhijit DASGUPTA
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-06-22
Filing date: 2017-06-22
Publication date: 2017-12-28
Anticipated expiration: 2018-12-22

Abstract

The present disclosure relates to high affinity nucleic acid aptamers for selectively capturing and detecting microbial, e.g., bacterial, contamination in a sample, e.g., water system. The present invention further relates to aptamer-based biosensors (aptasensors), including systems comprising aptasensors, e.g., portable systems, handheld systems, engineered for use in the context of smartphone and cloud data capturing for rapid and accurate detection of microbial contamination in a sample.

Description

APTAMER-BASED COMPOSITIONS AND METHODS FOR EXTRACTION AND DETECTION OF MICROBIAL CONTAMINATION

BACKGROUND

[0001] Water contamination from microbes, particularly bacteria, is a major public health issue across the globe. Recent reports of identification of flesh-eating bacteria in public water, Salmonella and Listeria contamination in foods, and E. coli in public waters or foods has raised public awareness of the need for better, more rapid and effective solutions. In particular, bacterial contamination by Salmonella causes more hospitalizations and deaths than any other type of germ/microbe found in food, and $365 million in direct medical costs each year. Recent studies suggest that health agencies investigating Salmonella illnesses should consider untreated surface water as a possible source of contamination.

[0002] The extraction of bacteria from water remains a laboratory challenge, requiring multiple manual steps and analytical tests in a remote reference laboratory. Furthermore, the biochemical/molecular complexity of bacterial testing necessitates transportation of lab samples to a reference laboratory rather than being able to perform a rapid, handheld testing at the point-of-care (POC). However, at present, no portable, handheld device for bacteria testing is available to geo-tag data prior to securely transmitting the data to a mobile cloud system for analysis and geo-mapping at a central data center.

[0003] Rapid, on-site detection of Salmonella contamination in untreated surface water sources is an essential, but currently challenging aspect of effective water quality monitoring programs. In the absence of such technologies, the precise monitoring aquatic bacterial contamination of Salmonella in the surface water sources across the country will likely remain difficult. Thus, there is a critical need for developing new technologies capable of rapid monitoring of aquatic bacterial contamination, especially in remote areas where the access to equipped laboratories is limited. As discussed herein, aspects of the present invention address these unmet needs by providing effective tools for precise/accurate, rapid monitoring of spread of Salmonella bacteria in the environment, thus decreasing the incidence of public health issues related to Salmonella contamination. BRIEF SUMMARY

[0004] The present disclosure provides, inter alia, compositions, systems, devices, platforms, methods, and kits for extraction/separation/concentration, and qualitative and/or quantitative detection/identification of microbial, e.g., bacterial contamination in a sample, e.g., water sample/resource.

[0005] In one aspect, the present disclosure provides an aptamer-based sorbent composition for solid-phase extraction (SPE) to specifically capture and separate or pre- concentrate a biological molecule (e.g., microbe, e.g., bacteria) from a sample (e.g., water resource). In certain advantageous embodiments, the aptamer-based sorbent composition comprises a nucleic acid (e.g., DNA) aptamer having high affinity and specificity for Salmonella bacteria, e.g., Salmonella enterica serovar Paratyphi A). In certain embodiments, the nucleic acid molecule comprises the following sequence: 5'- ATG- GAC-GAA-TAT-CGT-CTC-CCA-GTG-AAT-TCA-GTC-GGA-CAGCG-3' [SEQ ID NO: l]. Nucleic acid sequences having at least 85% identity to SEQ ID NO: l are also within the scope of the present disclosure.

[0006] In another aspect, the present disclosure provides a method for aptamer-based sorbent for solid-phase extraction (SPE) and pre-concentration of bacteria, e.g., Salmonella, from a sample, e.g., water resource. In certain embodiments, the aptamer- based SPE sorbent is packed in a column or cartridge.

[0007] In another aspect, the present disclosure provides a method for extracting/separating or concentrating bacteria or specific bacteria (e.g., Salmonella species) from a sample (e.g., water sample) comprising:

(a) contacting the sample with an aptamer; and

(b) detecting the presence of bacteria,

wherein the aptamer comprises nucleic acid molecules (e.g., single stranded DNA/oligonucleotides) which are immobilized on a base material/solid support/sorbent.

[0008] In another aspect, the present disclosure provides a method of detecting or identifying the presence of bacteria or specific bacteria or detecting microbial contamination in a sample (e.g., water) comprising: (a) contacting the sample with an aptamer; and (b) detecting the presence of bacteria, wherein the aptamer comprises nucleic acid molecules (e.g., single stranded DNA/oligonucleotides) which are immobilized on a base material/solid support/sorbent. [0009] In another aspect, the instant disclosure provides a portable electrochemical aptamer-based biosensor device (aptasensor) for on-site (e.g., at the point-of-care (POC)) detection of a biological molecule (e.g., Salmonella bacteria) in a sample (e.g., water resource). In certain embodiments, the aptasensor is cartridge or chip based.

[0010] In another aspect, the present disclosure provides a method for fabrication of a portable electrochemical aptasensor device for on-site (e.g., at the point-of-care (POC)) detection of a biological molecule (e.g., Salmonella bacteria) in a sample (e.g., water resource). In certain embodiments the electrochemical sensing is performed on a cartridge/chip-based aptasensor.

[0011] In another aspect, the present disclosure provides a method of detecting or identifying the presence of bacteria or specific bacteria or detecting microbial contamination in a sample (e.g., water) comprising: (a) applying the sample onto an aptasensor device; (b) detecting the presence of bacteria, wherein the aptamer-based biosensor comprises electroactive-labeled nucleic acid (e.g., DNA) sequences immobilized on a 3-pixel carbon-printed electrode, and wherein an electrochemical signal is produced and recorded to allow for the detection.

[0012] In another aspect, the present disclosure provides a "signal off folding-based electrochemical aptasensor on a 3 -pixel gold-plated screen-printed carbon electrode for detection of aquatic bacterial contamination, e.g., Salmonella, in a sample (e.g., water resource).

[0013] In another aspect, the present disclosure provides a portable or handheld device, e.g., a smartphone controlled handheld device as a multi-channel potentiostat to control the electrochemical biosensors, perform/run electroanalytical experiments, and measure the current signals from biosensors, which is configured/adapted for use with any of the above-disclosed aspects of the present subject matter.

[0014] In another aspect, the present disclosure provides a mobile cloud system capable of managing geolocation tagging data and cloud database performing analytics displayed on a digital dashboard, which are engineered/configured/adapted for use with any of the above-disclosed aspects of the present subject matter.

[0015] In yet another aspect, the invention relates to a packaged article, e.g., an article of manufacture, such as a system, an assay and/or detection or diagnostic kit, comprising any of the components (e.g., aptamer-based sorbent composition; electrochemical aptasensor) of the invention, optionally with a label(s) and/or with instructions for use. Such label(s) include(s) components and/or compatible analytes. Such instructions include directing or promoting, including advertising, use of said article of manufacture.

[0016] In a further aspect, the invention relates to a method of manufacturing an article of manufacture comprising any of the components of the invention described herein, packaging the composition to obtain an article of manufacture and instructing, directing or promoting the use of the article of manufacture for any of the uses described herein. Such instructing, directing or promoting includes advertising.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0017] FIG. 1A-B show an exemplary aptamer-based SPE column for separation of

Salmonella bacteria. (A) Schematic representation of solid-phase extraction process of Salmonella bacteria using aptamer-modified silica nanoparticles (B) Salmonella bacteria extracted onto the extraction column can be transferred to remote labs or analyzed using on-site detection systems.

[0018] FIG. 2 shows a schematic representation of the structure of 3-pixel carbon-printed electrode and the corresponding DNA sequences used for modification of each channel electroactive-labeled DNA sequences which are used as sensing probes.

[0019] FIG. 3 shows a schematic representation of performance of different channels at

3-pixel sensor in the absence and presence of the target and their corresponding electrochemical signal.

DETAILED DESCRIPTION

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Additional definitions are set forth throughout the detailed description. [0021] Salmonella, as one of the most common pathogens of foodborne disease worldwide, is responsible for a large number of infections in both humans and animals. It is estimated that Salmonella causes 93.8 million human infections and 155,000 deaths annually worldwide. Efficient sample cleanup as a main part of each analysis is required to separate the target from its natural matrix, to pre-concentrate it, to improve the detection sensitivity of the analysis method, and to protect the detection device from possible damage. The most conventional cleanup method for Salmonella bacteria involves solid-phase extraction (SPE) with conventional cartridges or immune-affinity columns. However, conventional SPE methods require large volumes of organic solvent and are usually time-consuming. Also, immuno-affinity columns are expensive and can exhibit variability. Further, chaotropic salts, acidic or basic pH and organic solvents may cause irreversible denaturation of the antibodies, thus shortening immuno-affinity column life. To overcome these limitations, many efforts have been made to substitute conventional cartridges and immuno-affinity columns with low-mass synthetic ligands, aptamers and molecularly imprinted polymers.

[0022] The present disclosure provides, inter alia, compositions, systems, devices, platforms, methods, and kits for extraction/separation/concentration, and qualitative and/or quantitative detection/identification of microbial contamination, e.g., bacterial contamination, in a sample, e.g., water sample/resource, or for food sanitation, or medical diagnosis.

[0023] In certain aspects, the present disclosure provides technologies, e.g., compositions, methods, systems, devices for monitoring water quality. The increase in the number of recent reports of contamination of surface waters with Salmonella bacteria underscores the critical importance of the new technologies disclosed herein for detection of sources of water contamination. The present disclosure provides effective aptamer- based techniques and aptasensing technologies, including methods for engineering these technologies for implementation in combination with established electronic and computer technologies to develop practical methods for on-site separation and detection of aquatic bacterial contamination of Salmonella.

[0024] The present disclosure provides technologies in the field of sensors for fabricating cost-effective practical chemical-and biochemical (bio) sensors. In certain aspects, the present disclosure provides aptamer-based techniques for both extraction and detection of Salmonella in water resources. "Aptamers" are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), peptide nucleic acid (PNA) and any combinations thereof, that can bind to pre-selected targets with high affinity and specificity. Applicants utilized the strong, specific interaction between Salmonella bacteria to engineer tools comprising aptamers as capture agents to develop efficient separation and effective detection techniques, which allow for on-site separation and measurement of Salmonella bacteria in water sources. Applicants show that using such a specific interaction in the context of electrochemical aptasensors and its combination with recent technologies enables fabrication of a cost-effective, reliable Salmonella bacteria sensor.

[0025] In one aspect the present disclosure provides an aptamer composition having high affinity and specificity for Salmonella bacteria (herein referred to as "Salmonella aptamer") as a capturing agent for fabrication of solid-phase extraction column of Salmonella bacteria. In another aspect the present disclosure provides an electrochemical "signal-off aptasensor for Salmonella bacteria detection in which the Salmonella aptamer is used as a recognition element, and methods for design and fabrication thereof. In a further aspect, the present disclosure provides a sensor platform which effectively addresses the problems associated with conventional electrochemical "signal-off aptasensors.

[0026] In certain aspects, the present disclosure provides a portable, electrochemical aptasensor for on-site detection of Salmonella bacteria. The present disclosure provides an end-to-end solution because data from the potentiostat can be geo-tagged and sent to a mobile cloud system providing a real-time display of the geo-mapping results from point of care water tests.

Aptamers and aptamer -based sorbents for solid phase extraction (SPE):

[0027] In certain aspects, the aptamer-based techniques disclosed herein provide an aptamer-based sorbent for SPE using a SPE column or cartridge or chip for efficient, separation or pre-concentration of Salmonella bacteria from water samples. As such, any sorbent, solid support or base material may be used. As used herein, "base material," "sorbent," "solid support," "support," and "substrate" refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In some aspects, at least one surface of the solid support will be substantially flat, although in some aspects it may be desirable to physically separate synthesis regions for different molecules with, for example, wells, raised regions, pins, etched trenches, or the like. In certain aspects, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. In certain embodiments, the SPEs are metal-plated SPEs, e.g., gold- plated SPE. In certain embodiments, silica nanoparticles are used as the base material/sorbent for immobilization of aptamer onto it. Immobilization onto silica nanoparticles may be performed using known "click chemistry," or other methods. Additional non-limiting examples of sorbents and modification methods for immobilization of the aptamers, which may be used to practice the methods disclosed herein include, CarboxyLink Coupling gel (immobilized diaminodipropylamine, DADPA) as the base materials for immobilization of the aptamer, and the NHS/EDC coupling chemistry.

[0028] It should be appreciated that aptamers specific for any microbe (bacteria), including different species, serovars, etc. thereof, are within the scope of the present disclosure. Antibiotic-resistant Salmonella enterica serovar Paratyphi A (S. Paratyphi A), is chosen as a model for this study. However, aptamers directed to any serovar of Salmonella enterica are within the scope of the present disclosure.

[0029] Aspects of the present disclosure provide an exemplary aptamer with high affinity and specificity against Salmonella enterica serovar Paratyphi A (S. Paratyphi A) ("Salmonella A aptamer") having the following sequence:

5'- ATG-GAC-GAA-TAT-CGT-CTC-CCA-GTG-AAT-TCA-GTC-GGA- CAGCG-3' [SEQ ID NO: l]

[0030] Aspects of the present disclosure also provide aptamers having nucleic acid sequences having at least 85%, 87%, 90%, 95% or 100% identity to SEQ ID NO: l .

[0031] In certain embodiments, the Salmonella A aptamer is modified at the 5' end and/or at the 3' end. An exemplary modification at the 3' end is: - (CH2)5 NH2. An exemplary modification at the 5 'end is a thiol group. In certain embodiments, the Salmonella A aptamer is modified to include an electroactive group (electrochemical label) of methylene blue at the 3' end.

[0032] Aptamers within the scope of the present disclosure may include a detectable label attached thereto. The detectable label may be a moiety that may be detected by detection methods known in the art. For example, the detectable marker may be an optical label, an electrochemical label, a radioisotope or combinations thereof. The label may be attached to a certain base or certain structure of an aptamer, for example, a certain site of a hairpin- loop structure or a 3' end or a 5' end of an aptamer.

[0033] The optical label may be, for example, a fluorescent substance. The fluorescent substance may be chosen from the group consisting of fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, Comarin, Cy2 (cyanine 2), Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin, PerCP (peridinine chlorophyl-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'- dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor, 7-amino-4-methylcomarin-3-acetate, BODIPY FL, BODIPY FL-Br 2, BODIPY 530/550, their conjugates and mixture thereof. For example, the fluorescent substance may be fluorescein, Cy3 or Cy5.

[0034] Also, the optical label may be a pair of fluorescence resonance energy transfers

(FRET) that include donor fluorophore and acceptor fluorophore separated by an appropriate distance in which the fluorescence of the donor is inhibited by the acceptor. The donor fluorophore may include FAM, TAMRA, VIC, JOE, Cy3, Cy5 and Texas Red. The acceptor fluorophore may be selected such that excitation spectrum thereof overlaps emission spectrum of the donor. Also, the acceptor may be a non-fluorescent acceptor that quenches donors of broad range. Other examples of donor-acceptor FRET pair are known in the art.

[0035] An exemplary, non-limiting method for immobilizing the aptamer onto SPE base materials to form aptamer-modified SPE column is described below.

[0036] Briefly, silica nanoparticles are used as the base material for immobilization of aptamer. The immobilization of aptamer is performed using well-established "click" chemistry. SPE columns are packed with aptamer-modified sorbent containing fritted polyethylene diskettes on the bottom. The sorbent is allowed to settle, then another frit is placed on the gel top. Methods for efficient separation and extraction of Salmonella enterica servovar Paratyphi A (S. Paratyphi A), the aptamer-based SPE column are optimized with regards to different steps, including: 1) preparation and conditioning of the SPE column (proper conjugation chemistry, column condition optimization, including pH, ionic strength and composition of washing solution and elusion solution); 2) analytical merits of the SPE column performance including loading capacity, specificity toward separation of Salmonella enter ica servovar Paratyphi A (S. Paratyphi A) pre- concentration factors, regeneration and reusability, lifetime, and shelf life; and 3) applicability of the fabricated SPE column to separate Salmonella S. Paratyphi A from field samples is studied.

Folding-based electrochemical aptasensors:

[0037] Folding-based electrochemical aptasensors have gained substantial popularity, owing to the merits associated with this sensing platform. Most sensors of this class are essentially reagentless, rapid, reusable, sensitive, specific, selective, and are highly compatible with microfluidic-based detection platforms. The electrochemical sensor disclosed herein is based on a folding-based electrochemical biosensing platform developed at the University of Nebraska at Lincoln. A key feature of the electrochemical aptasensor-based bacterial detection system disclosed herein is the strategic modification of the reagentless and versatile sensing platform for on-site detection of aquatic bacterial contamination of Salmonella.

[0038] The sensing mechanism provided by the aptasensor disclosed herein exploits specific interactions between the Salmonella bacteria and its specific aptamer, and employs a "signal-off mechanism. The Salmonella A aptamer (with the sequence of 5'- ATG-GAC-GAA-TAT-CGT-CTC-CCA-GTG-AAT-TCA-GTC-GGA-CAGCG-3') is chosen as the recognition aptamer. Briefly, the method comprises the following steps: (a) carbon printed electrodes are modified with gold; b) Salmonella A aptamer (modified with thiol group at 5'end and electroactive group of methylene blue at 3'end) is immobilized on gold-modified carbon-printed electrodes through thiol chemistry; c) the electrode surface is further passivated with alkanethiols; d) the electrochemical signal of methylene blue before and after binding to Salmonella A aptamer is recorded.

[0039] In certain aspects, the present disclosure provides a method for fabrication of a folding-based electrochemical aptasensor for detection of Salmonella A in water resources.

Electrochemical sensor platform:

[0040] In certain aspects, the present disclosure provides an electrochemical aptasensor platform which overcomes the problems associated with conventional electrochemical sensors. The most conventional functional mode of folding-based electrochemical aptasensors is "signal-off mode. However, this mode has three main limitations. First, this mode suffers from false positive responses. Such false positive responses are due to different unknown, uncontrollable factors present in the matrix of real samples, which affects senor performance in an unknown manner. In this respect, poor quality of reagents and sensor performance during the experiment might affect the reliability of the obtained results as well. Thus, examining the validation the obtained results is of prime importance in order to confirm the credibility of obtained results. Second, performing calibration experiments prior to each analysis could deteriorate the sensor structure rendering the obtained data less reliable. Thus, the quality of reagents used for each analysis needs to be checked constantly, preferably prior and within each analysis. The aforementioned factors make use of "signal-off sensors less straightforward for the on- site applications. Third, considering the fact that most of fabricated aptasensors work with "signal-off mode, and designing "signal-on" mode is not feasible for most of them, it is clear that optimizing of "signal-off sensor will greatly enhance the application of this type of sensor, paving the way for on-site application of this type of sensors. The exemplary assay discussed below addresses the aforementioned problems through incorporating a validating and self-calibrating mechanism into each sensor structure.

[0041] Figure 2 schematically shows the structure of the proposed sensor platform and used reagents 1) as shown in Figure 2, two following DNA sequences, which are used as signal producing probes, will be synthesized and mixed with same ratio:

[0042] 5'- Methylene blue (MB) - Control sequence composed of four T nucleotides

(TTTT) - Salmonella aptamer (Apt) - 3'— : (MB-TTTT-Apt)

[0043] 5'- Methylene blue (MB) -CCCC) - random DNA sequence composed of same number of nucleotides as Apt (Random) - 3' -^π: (MB-CCCC-Random)

[0044] 2) 3 -pixel carbon-printed electrode is modified with gold, and each pixel (channel) are is modified with DNA sequences through thiol chemistry:

Channel#l AAAA (complementary sequence to control probe)

Channel#2 Random Comp (complementary sequence to Random)

Channel#3 Apt Comp (complementary sequence to Apt)

[0045] 3) The mechanism of sensor performance in the absence and presence of target i.e.

Salmonella, can be described as follows (as shown in Figure 3): [0046] A) In the absence target:

[0047] Channel # 1 : The hybridization between AAAA probe, immobilized on electrode surface, and its complementary portion on MB-TTTT-Apt probe will place the MB close to the surface of electrode producing an electrochemical signal. The signal at channel # 1 is used as a positive control signal to confirm the viability of MB-TTTT-Apt probe in the test environment and to validate the experiment.

[0048] Channel #2: The hybridization between Random Comp, immobilized on electrode surface, and its complementary portion on MB-CCCC-Random will place the MB close to the surface of electrode producing an electrochemical signal. Such hybridization is not affected by the presence or absence of the target. However, since the structure, length, and concentration of MB-CCCC-Random is similar to MB-TTTT-Apt, the electrochemical signal at channel#2 provides a reference signal showing the expected amplitude of signal of a blank sample at sensor surface at each step of experiment. Thus, signal at channel # 2, ideally should be constant within an experiment and also between different experiments.

[0049] Channel #3: The strong interaction between Apt portion on MB-TTTT-Apt probe and the target decreases its hybridization efficiency with Apt Comp sequence, immobilized on channel#3, thus, decreasing the electrochemical signal recorded at channel #3. The difference in the signal amplitude with respect to channel#2 is considered as the amplitude of the sensor response to target.

[0050] For real sample analysis, other scenarios are also possible. The major scenarios and their corresponding interpretations are listed in Table 1. As Table 1 shows each possible situation can be used to validate the response of "signal-off ' sensor.

TABLE 1 : Possible scenarios and corresponding signal interpretation of each situation:

Channel#

Condition Results

1 2 3

No Signal Invalid

A No Signal Invalid

If C»B

Electrochemical

A B C Invalid Signal

Or C»A A B C B>C Valid

[0051] There is a possibility that hybridization efficiency of MB-TTTT-Apt will not be the same with two different complementary probes of AAAA and Apt Comp immobilized on channel# l and channel#3 respectively. Thus, the electrochemical signal at these two channels would not be the same in the absence of target. At this situation, the absolute difference in electrochemical signal of channel# l and channel#3 still can be used as a tool as positive control experiment.

Multi-channel potentiostat and smartphone controlled handheld device:

[0052] In certain aspects, the present disclosure provides a method for designing a smartphone-controlled handheld device with appropriate hardware engineering and firmware engineering. A multi-channel potentiostat is the required electronic component to control the electrochemical biosensors, run electroanalytical experiments, and measure the current signals from biosensors. Most of the potentiostat equipment available on market is costly and bulky, which limits portability and application of such electrochemical analysis systems. Although there are a few handheld potentiostat products, they are still very expensive (costs more than $10,000), while only have limited capabilities that cannot meet the requirements of the electrochemical biosensors and system. To address/solve these challenges, the present disclosure provides a customized potentiostat for the electrochemical biosensors disclosed herein, using state-of-the-art electronic devices to satisfy the needs while minimizing cost (less than $500) and size. The potentiostat circuit is integrated into a handheld embedded system with low-power microprocessors and Bluetooth low energy (BLE) device, which works with a smartphone in the form of an accessory device. This design will not only accelerate the research and development progress, but also provide users with cost-effective reliable solutions for collecting geographical information and accessing data networks, by utilizing widely- available smartphones. In terms of hardware engineering, an industry standard process flow is utilized while customized for the electrochemical biosensor. Briefly, the industry work flow standard includes: 1) enumerating the requirements of the handheld device; 2) designing and generating hardware specifications based on the requirements; 3) designing printed electronic circuit boards (PCB) using CadSoft Eagle PCB or Cadence OrCAD Design tools; 3) simulating the design to verify that the design satisfies both the specifications and the requirements of 3rd party fabrication houses; 4) generating bill of materials (BoM) based on the PCB design and selected components; 5) constructing prototypes of the handheld device; 6) making a test plan to verify achievement of specifications; 7) developing appropriate quality test plans for manufactured handheld devices.

[0053] The hardware design highlights include a low-power microprocessor; single- channel or multi-channel 12-bit or 16-bit Digital -to- Analog Converters (DAC); a sine wave generator; multi-channel 12-bit or 16-bit Analog-to-Digital Converters (ADC); customized multi-channel potentiostat circuits capable of performing various types of voltammetry; Bluetooth low energy (BLE) communication module; rechargeable battery, power management circuits and charging station; user interfaces.

[0054] In terms of firmware engineering, a bioengineer architects, implements and tests the firmware to be running on the smartphone controlled handheld device. The bioengineer will perform the steps of: 1) creating the firmware specifications and use cases to meet the requirements of the handheld device; 2) designing the architecture of the firmware; 3) designing and implementing communication protocol for information exchange between the handheld device and smartphones; 4) implementing algorithms to achieve various types of voltammetry as listed in the firmware specifications; 5) designing and implementing functions coordinating all electronic components in the system; 6) testing and documenting the firmware source codes.

[0055] In further aspects, the present disclosure provides methods for designing and developing a smartphone app. A smartphone app is required to control the operations of the handheld device and electrochemical biosensors. It is the essential software that processes and stores the collected data, including electrochemical tests data and results, geographical information, and other useful information. The data is synchronized with a dedicated cloud server for further processing, data analysis, and other value-added services.

[0056] The app disclosed herein provides users with a graphical user interface (GUI) to:

1) control and monitor the status of the handheld device; 2) store the test results acquired from the electrochemical biosensor; 3) visually present the results to the users; 4) record the geographic information using the geographic information system (GIS) applications embedded in the smartphone; 5) perform necessary algorithms for signal processing and interpretation; and 6) transmit the data through Wi-Fi network or mobile network to the cloud server. In addition, an advanced encryption algorithm is used in data storage and transmission to assure information security. As the major user interface, it is very important to keep the app simple and easy-to-use without sacrificing the functionalities.

[0057] Designing and implementing the smartphone app are achieved by: 1) enumerating/creating the requirements of the smartphone app; 2) designing and documenting the graphical user interface (GUI) and processes of the smartphone app; 3) implementing the smartphone app according to the requirements and the designs; 4) implementing an in-house Bluetooth communication protocol; 5) implementing algorithms designed in-house for signal processing and interpretation; and 6) testing and documenting the smartphone app.

Mobile cloud system:

[0058] In one aspect, the present disclosure provides a mobile cloud system that can handle geo-tagged data from the handheld potentiostat and be received by a central command data center for geo-mapping and analytics. The following steps are developed into the components and system:

• Communication powered by cell phone system or radio, not Bluetooth (too far from base or rest of world, potentially

• Geolocation tagging using GPS chip

• Transmit data with geolocation to cloud base

• Dashboard can visualize different tests on a map along with reading levels taken with product. This is easy using geolocation, Leaflet.js, Google Maps interfaces

• Cloud database will store geotagged readings for current delivery as well as later spatial analyses. Can do things like :

o Looking at pollution flows in real time

o Look at geospatial patterns of biological, organic and inorganic pollutants o Allows early warning of public health hazards during emergencies, by monitoring upstream

o Quick residential water testing after known contamination to determine safety • Technologically:

o Existing cloud database will suffice for storage.

o Determine how cellular or radio networks will interface with Internet based infrastructure. Determine the interface points be with the mobile cloud, o Design of dashboard uses central theme of location rather than readings.

^■ Central map with pull-downs or buttons with different options for reading choices or the like

^■ Zoom in or out via fingers or mouse

^■ Main mode of delivery will be iPad or 7-10 inch screens, not phones

^■ Maybe some color coding

^■ Recruit domain knowledge of what to look for here.

• Analytics include, for example,

o GIS (Geographical Information Systems); open source available aplenty o Geostatistical analysis

o Flow analyses, linking with GIS topography

o Linking with news feeds, Twitter/FB, FEMA to associate data with real-time events

o US Geological Survey good resource for maps, as is Google Earth and competitors.

[0059] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0060] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an aptamer-based sorbent for solid-phase extraction (SPE) for specifically capturing/separating or pre-concentrating bacteria from a water sample.

2. The composition of claim 1, wherein the aptamer-based sorbent comprises silica nanoparticles.

3. The composition of claim 1, wherein the aptamer-based sorbent comprises a nucleic acid aptamer which is immobilized on the sorbent.

4. The composition of claim 3, wherein the aptamer is a deoxyribonucleic acid (DNA) molecule having high specificity and affinity for Salmonella bacterial species, e.g., Salmonella enterica serovar Paratyphi A.

5. The composition of claim 4, wherein the DNA molecule comprises a sequence having at least 85% identity to the following sequence: 5'- ATG-GAC-GAA-TAT-CGT-CTC- CCA-GTG-AAT-TCA-GTC-GGA-CAGCG-3' [SEQ ID NO: l].

6. The composition of any one of claims 3-5, wherein the aptamer comprises a modified/functional group or a detectable label.

7. The composition of claim 6, wherein the detectable label is an electrochemical/electroactive label.

8. A solid phase extraction (SPE) column for capturing/separating or pre-concentrating bacteria from a water sample comprising aptamer-modified silica nanoparticles, wherein the aptamer comprises deoxyribonucleic acid (DNA) molecules having high specificity and affinity for Salmonella bacterial species, e.g., Salmonella enterica serovar Paratyphi A.

9. The column of claim 8, wherein the DNA molecules comprise a sequence having at least 85% identity to the following sequence: 5'- ATG-GAC-GAA-TAT-CGT-CTC-CCA- GTG-AAT-TCA-GTC-GGA-CAGCG-3' [SEQ ID NO: l].

10. A method for extracting/separating or concentrating bacteria or specific bacteria (e.g., Salmonella species) from a sample (e.g., water sample) comprising:

(a) contacting the sample with an aptamer; and

(b) detecting the presence of bacteria,

11. The method of claim 10, wherein the DNA molecules comprise a sequence having at least 85% identity to the following sequence: 5'- ATG-GAC-GAA-TAT-CGT-CTC-CCA- GTG-AAT-TCA-GTC-GGA-CAGCG-3' [SEQ ID NO: l].

12. A method of detecting or identifying the presence of bacteria or specific bacteria or detecting microbial contamination in a sample (e.g., water) comprising:

(a) contacting the sample with an aptamer; and

(b) detecting the presence of bacteria,

13. The method of claim 12, wherein the DNA molecules comprise a sequence having at least 85% identity to the following sequence: 5'- ATG-GAC-GAA-TAT-CGT-CTC-CCA- GTG-AAT-TCA-GTC-GGA-CAGCG-3' [SEQ ID NO: l].

14. A portable electrochemical aptamer-based biosensor device (aptasensor) comprising nucleic acid aptamers, for on-site (e.g., at the point-of-care (POC)) detection of a biological molecule (e.g., Salmonella bacteria) in a sample (e.g., water resource).

15. The aptasensor of claim 14, wherein the aptasensor is cartridge-based or chip-based.

16. The aptasensor of claim 14, wherein the aptasensor is a "signal off folding-based aptasensor.

17. The aptasensor of claim 16 comprising a 3-pixel gold-plated screen-printed carbon electrode.

18. The aptasensor of claim 17 comprising electrochemically/electroactively-labeled nucleic acid (e.g., DNA) sequences immobilized on the 3-pixel carbon-printed electrode.

19. The aptasensor of claim 14, wherein the aptamers comprise a nucleic acid sequence having at least 85% identity to the following sequence: 5'- ATG-GAC -GAA-T AT-C GT- CTC-CCA-GTG-AAT-TCA-GTC-GGA-CAGCG-3' [SEQ ID NO: l].

20. The aptasensor of claim 19, wherein the aptamer is modified with a thiol group at the 5 'end and an electroactive group of methylene group at the 3 'end.

21. A method for fabrication of a portable electrochemical aptasensor device for on-site (e.g., at the point-of-care (POC)) detection of a biological molecule (e.g., Salmonella bacteria) in a sample (e.g., water resource), the method comprising the steps of:

(a) modifying carbon printed electrodes with gold;

(b) immobilizing a Salmonella A aptamer (modified with thiol group at 5 'end and electroactive group of methylene blue at 3 'end) on the gold-modified carbon-printed electrodes of step (a) using thiol chemistry;

(c) passivating the electrode surface with alkanethiols; and

(d) recording the electrochemical signal of methylene blue before and after binding to the Salmonella A aptamer

22. A method of detecting or identifying the presence of bacteria or specific bacteria or detecting microbial contamination in a sample (e.g., water) comprising:

(a) applying the sample onto an aptasensor device;

(b) detecting the presence of bacteria, wherein the aptamer-based biosensor comprises electroactive-labeled nucleic acid (e. DNA) sequences immobilized on a 3-pixel carbon-printed electrode, and wherein electrochemical signal is produced and recorded to allow for the detection.

23. A mobile cloud system configured for receiving geo-tagged data from a handheld potentiostat, wherein the geo-tagged data is received by a central command data center for geo-mapping and analytics.