PCR Technologies for Point of Care Testing ... - ACS Publications

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PCR Technologies for Point of Care Testing: Progress and Perspectives Salvatore Petralia*,† and Sabrina Conoci*,† †

STMicroelectronics, Stradale Primosole 50, 95121 Catania, Italy ABSTRACT: Since the Human Genome Project completed in 2000, the sequencing of the first genome, massive progress has been made by medical science in the early diagnosis and personalized therapies based on nucleic acids (NA) analysis. To allow the extensive use of these molecular methods in medical practice, scientific research is nowadays strongly focusing on the development of new miniaturized and easy-to-use technologies and devices allowing fast and low cost NA analysis in decentralized environments. It is now the era of socalled genetic “Point-of-Care” (PoC). These systems must integrate and automate all steps necessary for molecular analysis such as sample preparation (extraction and purification of NA) and detection based on PCR (Polymerase Chain Reaction) technology in order to perform, by unskilled personnel, in vitro genetic analysis near the patient (in hospital, in the physician office, clinic, or home), with rapid answers and low cost. In this review, the recent advances in genetic PoC technologies are discussed, including the extraction and PCR amplification chemistry suitable for PoC use and the new frontiers of research in this field. KEYWORDS: PCR, nucleic acids, PoC, sample prep, lab-on-chips, electrical transduction, intercalating agents, labeled-probes, fully integrated systems

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ince 2000 when the Human Genome Project completed the sequencing of the first genome, tremendous progress has been made by medical science in the early diagnosis and personalized therapies based on nucleic acids analysis. Nowadays the sequence of thousands of genomes is known and this number has enormously increased in recent years. According to data reported in the GeneBank database1 the sequencing of about 2350 viruses, 14 800 bacteria, and 2100 eukaryote organisms (included our species, Homo sapiens) (Figure 1) has been completed, with active movement in this direction achieved in the last five years. This has impressively enabled progress in many fields including anthropology, forensic science, and, in particular, medicine. In fact, the molecular analysis of nucleic acids (DNA, RNA) has stimulated truly disruptive innovation in the healthcare area that is effective in early diagnosis, personalized therapy, and preventive cancer screening, helping healthcare professionals to prescribe accurate therapeutic interventions. This has opened innovative medical perspectives in many fields of medicine including infectious diseases,2 oncology,3 pharmacogenomics,4 genetic diseases,5 diabetes,6 forensic science,7 and neurological8 and cardiovascular diseases.9 From the analytical point of view, the analysis of DNA sequences can address two main targets: (i) the human genomic DNA and (ii) virus/bacteria (DNA or RNA) genomes. The former is mainly involved in genetic diseases5 and personalized therapies,4 while the latter are particularly relevant in the diagnosis of infectious diseases. © XXXX American Chemical Society

Figure 1. Number of sequenced genomes submitted to NCBI for Virus (green bar), Bacteria (blue bar), and Eukaryotic kingdoms (yellow bar). Data source: ref 1.

The invention and development of polymerase chain reaction (PCR) technology10 has created a revolution in nucleic acids (NA) analysis, expanding it to large laboratory scale (Kary B. Mullis, who invented it, was awarded the Nobel Received: May 5, 2017 Accepted: July 6, 2017 Published: July 6, 2017 A

DOI: 10.1021/acssensors.7b00299 ACS Sens. XXXX, XXX, XXX−XXX

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risk of pandemics due to the re-emergence of infectious diseases from globalization17 and current uncontrolled migration flows. Much more important, the potential utility of these devices is in developing countries, where clinical laboratory infrastructures are very poor and cost constraints are relevant. In these countries infectious diseases are causes of mortality and morbidity for more than half of all infant deaths.18,19 In fact, the development of PoC technologies for diagnosing infectious diseases has been identified by the Bill and Melinda Gates Foundation and the National Institute of Health (NIH) as one of the major priorities in the “Grand Challenges for Global Health”.20 In view of these perspectives, the molecular diagnostics market analysts see genetic PoC as one of the most important sectors for the growth of this entire molecular diagnostics market segment up to a forecast of about 8 billion USD in 2024.21 In this review we discuss the recent advances in genetic PoC technologies. In particular, after the description of the fundamentals of the chemistry for NA extraction (sample preparation) and detection (amplification technologies), we will focus on the currently available fully integrated technological solutions for genetic PoC systems, including some commercial products that have recently appeared in the market. Finally, we will illustrate the new frontiers of the research in this field.

Prize in Chemistry in 1993). This method is based on the amplification of a specific genetic sequence, unique to an individual organism, through thermal cycling by the catalytic action of the polymerase enzyme. This technology has been extensively studied and optimized over the past years with a plethora of new variants (isothermal PCR, real-time PCR, and so forth).11,12 However, to execute NA molecular analysis several analytical steps need to be accomplished. First of all, (a) NA extraction from a biological samples, i.e., blood, urine, saliva, swab (sample prep); then, (b) target sequence amplification (PCR); and finally, (c) detection of the amplified product (Figure 2a).

Figure 2. Schematic illustration of NA analysis: (a) current laboratory steps and (b) PoC format.

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Thanks to the progress made in the optimization of the PCR method, steps (b) and (c) can now be merged with so-called real-time PCR able to amplify, detect, and quantify the target sequence in a single step.13 This method can be considered a further pillar in the NA analysis since it allowed the PCR methodology to enter into the diagnostic field avoiding any type of amplicon contamination and preventing false positive results.14 The execution of an NA molecular test is still quite laborious and time-consuming. It involves several manual steps including reagent dilution and mix preparation. Therefore, it requires specialized instrumentation and personnel. Additionally, it is still quite expensive ($20−80 per sample) so that nowadays it is exclusively performed in specialized centralized laboratories (Figure 2a). To allow the extensive use of these molecular methods in medical practice, great efforts have been made from researchers to develop easy-to-use miniaturized technologies and devices allowing NA analysis to be executed by unskilled personnel with sensitivity comparable to PCR technologies (LoD 10 copies/reaction) at competitive cost (few dollars per test).15,16 These systems are called Genetic “Point-of-Care” (PoC) and are designed to be used in decentralized environments (outside the central laboratory) near the patient. From the technological point of view they must integrate the three steps necessary for molecular analysis as illustrated in Figure 2b. To achieve this goal, technological advancements play a pivotal role. Several multidisciplinary competencies are needed ranging from molecular biology and chemistry to optical and electrical engineering, microfluidic and software algorithms. In the medical field, the impact of these types of devices will be massive. In the developed world these technologies will allow substantial diagnostic screening, making important advancements in all the clinical fields mentioned earlier, allowing direct timely therapeutic interventions and improving patients’ clinical outcomes. Additionally they will help in the

SAMPLE PREPARATION CHEMISTRY In molecular analysis, the sample preparation step is executed before PCR amplification. It is a bottleneck step because the quality (purity and integrity) of the purified NA is crucial to obtain meaningful and reproducible PCR data. This is, of course, particularly relevant in PoC systems where volumes and analytical steps are often reduced. Standard sample preparation methods are based on the following steps: (i) cell lysis, (ii) NA binding on solid supports, (iii) washing, and (iv) NA elution (Figure 3a). These steps are executed in analytical laboratories using standard methods such as mechanical/chemical lysis, centrifugation, solvent extraction,

Figure 3. Schematic illustration of sample preparation: (a) main steps; (b) prepackaged disposable filter-membrane format; (c) paramagnetic beads format; and (d) micropillars format. B


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(ii) Charge-based reversible adsorption (ion exchange chromatography). In this approach, charged NA molecules are selectively linked to solid surface through a charge exchange with the counterions placed at the surface. Thus, NA bind to the solid surface by charge and the unbounded molecules are washed away. (iii) Reversible surface absorption (af f inity chromatography). This method has been proven to be the most effective in the case of DNA and thus is commonly implemented in extraction kits. The core of this SPE mechanism is based on a specific binding occurring between the NA and the solid sorbent surface. In the case of sorbent material based on silicon oxide or tetraethyl orthosilicate (TEOS), the binding is due to hydrogen bond formation between the hydroxyl groups present on the surface and the DNA strands, in the presence of high concentration of chaotropic salts and at low pH values.37 In fact, in the presence of high ionic strength conditions, the water molecules absorbed on the negatively charged silica surface are removed to allowing the surface DNA binding.38 The further washing steps (typically carried out with specific washing buffers) remove the eventual interfering species absorbed on the silica surface. Finally, during the elution phase, the bounded DNA is released from surface through a surface hydration mechanism.39 In fact, elution is usually performed by aqueous solutions and no specific chemicals are required. The above-described SPE procedure can be implemented in different formats providing the sorbent silicon oxide surface in the following: (i) Prepackaged disposable f ilter membrane22 (Figure 3b). This format has the advantages of good specificity for DNA extraction and low cost. The main drawbacks are related to the need for many reagents (extraction, elution, washing buffers) requiring multiple microfluidic steps with limited portability into PoC systems. (ii) Paramagnetic beads23 (Figure 3c). This format offers the advantage in possible bead surface derivatization with specific probes, making it very effective in NAs isolation with moderate cost. Again, many reagents are required with consequent complex instrumentation. (iii) Microstructures like micropillars40 (Figure 3d). This format is featured by high surface-to-volume ratio inducing high extraction efficiency. It is very suitable for integration into miniaturized systems. These three SPE formats, coupled with specific lysis approaches, could be properly integrated into PoC systems generating devices with various levels of miniaturization. For example, the GeneXpert (Cepheid) system integrates a mechanical lysis by sonication with the nucleic acid separation method based on surface affinity, in a microfluidics plastic device of some centimeters (see the following section). Also, the FilmArray (BioMerieux) system integrates the mechanical lysis by bead milling with the silica magnetic bead separation in a plastic cartridge of some tens of centimeters (see the following section). In some cases featured by specific applications and biological specimens, the sample preparation can be performed in a single-step. An interesting example that uses this approach is reported in our previous work for the detection of β-globin gene directly from human blood.41 We demonstrated the successful integration of the enzymatic lysis (performed by a protease based reagent developed by ZyGem) on silicon microfluidic lab-on-chip followed by DNA amplification and

or precipitation depending of the extraction kit/method used.22−24 Although these methodologies allow high-quality extracted NA to be obtained that are appropriate for downstream detection by amplification, few of them are suitable for miniaturization and integration in a PoC systems. In fact, to be considered for further PoC integration the extraction method should feature few analytical steps, flexibility (suitable for more type of samples), fast, low cost, and miniaturizable. In the following section we will review the chemistry associated with the main analytical steps at the basis of the NA extraction and purification (Figure 3a) and the related technologies (solid phase extraction, magnetic beads, and so forth), with particular emphasis on those able to be integrated into PoC systems. Cell Lysis. The release of NA from human cells, nuclei, and bacterial and viral capsids is essential for the sample preparation process. The methods employed are strongly dependent on the starting sample materials. Cell lysis for many cells (human cells or some pathogen species) can be performed using chemical agents (chemical lysis), like alkaline solution,25 salts,26 or chaotropic or detergent species.27 These agents are low cost and active already at room temperature. Thus, they require instrumentation with low complexity and low power consumption. However, they can create possible PCR interference; therefore after lysis with these agents, a NA purification step is needed. Finally, they are not very effective in some cases such as in the presence of hard-to-open cells (e.g., spores and fungi). Certain microorganisms need the use of some lytic enzymes such as the lysozymes, proteinase-K, or mutanolysin (enzymatic lysis).28 These enzymes have the characteristics to digest the peptidoglycan layer and membrane-protein degrading the membrane or capsid protein. In this context several companies such as Zygem29 and Ivagen30 have developed protease-based kit reagents for the rapid nucleic acid lysis and extraction of genomic, viral, and bacterial DNA/or RNA from biological samples. Although these technologies require specific enzymatic temperature and protein inactivation/digestion, they can be easily integrated in a PoC system, because they require a simple microreactor (made of plastic, silicon, or glass material) externally heated to perform the isothermal enzymatic reaction. Some hard-to-open pathogen species such as fungi, spores, and oocysts having a complex outer membrane are resistant to the common chemical and enzymatic lyses agents. These organisms require physical lysis mainly based on heat,31 pressure,32 sonication with sound waves,33 and bead milling.34 Although mechanical lysis requires complex instrumentation and large input of energy, it is a fast and flexible process that does not require the need of specific chemical agents or enzymes. Some of these are implemented in PoC systems such as Cepheid GeneXpert.35 DNA Extraction/Isolation. Once lysis is completed, the NA must be extracted and purified from the sample. The most suitable and miniaturizable NA separation technology is solidphase extraction (SPE). It allows an exhaustive removal of impurities from a flowing liquid sample via retention on a solid sorbent and subsequent recovery of NA target by elution from the sorbent.36 General SPE technology includes three main approaches: (i) Size exclusion (gel f iltration). In this case, the NA molecules are separated from other molecules by size through a gel matrix with a specific pore dimension. It allows NA to pass through, while the smaller molecules are held in the pores. C


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ACS Sensors Table 1. Sample Preparation Technologies Comparison sample preparation steps cell lysis

technologies chemical lysis

enzymatic lysis

mechanical lysis DNA extraction/ isolation

SPE - filtermembrane format SPE - paramagnetic beads format SPE - micropillars format

advantages

suitability versus PoC

disadvantages

active at room temperature low cost instrumentations with low complexity low power consumption very effective for cell lysis possible direct PCR amplification without DNA extraction/ isolation instrumentations with low complexity moderate power consumption fast, flexible and very effective for all cells including the hard-toopen cells (i.e., MTB, fungi, spores, etc.) good specificity low cost high specificity moderate cost high specificity and extraction efficiency miniaturization integration of microfluidic steps

possible PCR interference NA purification is needed not very effective for hard-to-open cells (i.e., MTB, fungi, spores, etc.)

+

specific temperature required possible PCR interference enzyme digestion or inactivation is needed

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complex instrumentations high power consumption many reagents and microfluidic steps are needed complex instrumentations many reagents and microfluidic steps are needed complex instrumentations moderate cost

++ +

++

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microarray detection on blood sample without further purification steps. This approach bases on protease enzyme ability to purify the DNA template by removing nucleoproteins and on its further thermal inactivation at the same temperature step (95 °C for 300 s) used for Taq polymerase activation. Table 1 summarizes the main advantages and disadvantages of the several sample preparation technologies discussed above and their suitability versus PoC integration.

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PCR AMPLIFICATION CHEMISTRY The Polymerase Chain Reaction (PCR), conceptualized and published for the first time by Mullis in 1985,10 has revolutionized the field of molecular biology. It consists on an exponential amplification of a specific NA target sequence thermally mediated by the polymerase enzyme and specific oligonucleotides primers. PCR allows the simultaneous amplification of multiple targets in parallel (multiplexing detection). This standard PCR methodology is based on three temperature steps: (1) DNA melting (denaturation step), (2) annealing, and (3) primers extension. In the original method, the detection of the amplified products was made after the reaction (end point PCR) using external methodologies such as agarose-gel electrophoresis,42 DNA hybridization,43−45 and enzyme-linked hybridization.46 Starting from that, in 1992 Higuchi introduced the renowned real-time PCR method.13 It merges amplification and detection permitting a real-time fluorescent monitoring of the target while its amplification is progressing. Thus, an amplification kinetic curve is obtained plotting the fluorescent signals versus the PCR cycle (see plotting reported in Figure 4a). It is also possible to calculate the target concentration (quantitative method) thanks to the correlation of the threshold cycle (Ct) in the kinetic curve and DNA concentration.47,48 Nonetheless, the instrument complexity, thanks to the ability to integrate the amplification with the detection step, the higher sensitivity and larger dynamic range with the possibility to perform multiplexing analysis, the real-time PCR technology is nowadays the

Figure 4. Overview on intercalative probe approaches in RT-PCR: (a) general scheme of intercalative agent; (b) mechanism of fluorescent intercalative probes; (c) mechanism of redox intercalative probes; (d) mechanism of intercalative redox solid-phase RT-PCR.

most used method in analytical laboratories for diagnostic applications. To improve the performance of this method researchers have developed a variety of approaches focused either on the chemistry of probe used for the detection (taqman probes, becon probes, and so forth) or on the thermal steps (two-step PCR and isothermal PCR - see infra). Additionally, PCR facilitator agents play a key role for the improvement of PCR efficiency in real-time methods, since they reduce the inhibitory effects. These three aspects are described in detail in the following sections. RT-PCR Probe Chemistries. These approaches are mainly based on the following categories: (a) double strand intercalative agents (first type), (b) labeled oligonucleotide probes (second type), (c) labeled primer-probes (third type), and, with minor incidence, (d) methods with the so-called “DNA-like agents” (PNA, LNA, ZNA probes) (fourth type).49 These methods mainly differ on the nature of the probe species (intercalating agents or labeled probes/primers) and their D


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The electrochemical detection offers numerous advantages such us facile integration in miniaturized electronic systems, good sensitivity, and robustness.55 An alternative innovative PCR technology based on intercalating agents is known as “solid phase PCR” (SPP). It consists of DNA amplification by elongation of primers covalently bound at the surface of a solid support, leading to a primer-extended product chemically linked to the surface (Figure 4d). The detection is based on intercalation of fluorescent or redox agents into the dsDNA directly linked to the electrode surface. In 2000, Adessi et al.56 published a detailed work on solid phase DNA amplification mechanisms. The authors demonstrated that there are two distinct mechanisms during SPP. In particular, the first is an “interfacial amplification” occurring when the DNA target repeatedly hybridizes with the attached primers and it is extended; the second, called “surface amplification”, proceeds upon the hybridization of surface linked extended DNA copies with attached primers forming a loop-closed system. Recently we have developed a solid phase real-time electrochemical PCR method for the detection of HBV Hepatitis-B virus in a miniaturized silicon device.57 5′-Thiolated primers (20-mer long) were chemically grafted on platinum working electrode surface. DNA amplification was detected in real time in the presence of redox intercalator agent (Os[(bpy)2DPPZ]+2 at a concentration of 0.1 μM. The square wave measurements showed the increasing of current density with the increases of PCR cycles number. The main advantages of this approach are certainly the high versatility and the low cost. In fact, thanks to the high specificity of the intercalation process into the double strand versus the single strand, this approach can be employed to detect various double helix DNA targets. Moreover, the signal coming from the intercalative agents is very flexible to be easily monitored by several transduction methods such us optical, electrochemical, and electric. Additionally, this approach is suitable for used on solid phase PCR, making it a very good candidate to be integrated on miniaturized low cost PoC devices. The disadvantages are mainly related to the unspecificity toward different double strand DNA targets that induces a severe DNA purification process. Labeled Probes (Second Type Probe). The labeled probes are oligonucleotides properly modified at their ends with a reporter fluorophore (R) and quencher molecule (Q) able to specifically hybridize with a portion of the amplified DNA (Figure 5a). Two are the main mechanisms employed for this type of probes: (i) hydrolysis or (ii) hybridization. The first mechanism is the most widespread and it is used in the well-known TaqMan probes.58 With this method the fluorescent signal is generated at the end of the extension phase through the release of R molecule mediated by the hydrolysis mechanism relies of the 5′-3′ exonuclease activity of Taq polymerase (Figure 5a). The second mechanism (hybridization probe) includes many approaches such as fluorescence resonant energy transfer (FRET)-probes (first described in 1985 by Heller and Morrison,59 molecular beacons probe (described for the first time by Tyagi and Kramer60 and the HybBeaconTM-probes (Figure 5b). All these methods are based on FRET from an attached-donor molecule (R) to an acceptor (Q), with these two moieties closed with each other. After the denaturation phase, these probes are hybridized on the PCR products and

interaction with the double strand DND (dsDNA) target. In particular, for the intercalant molecule (first type probe), the type of interaction with the target is nonspecific. On the contrary, in the case of labeled probe/primers (second and third type probes) the interaction is highly specific, being driven by complementary hybridization. The nature of the label molecule is critical for the transduction method. Typically the labels are compounds with fluorescent, redox, or bioluminescent properties. While the probe-target interaction influences the efficiency, sensitivity and specificity of the amplification, the labeling mode affects the transduction technology. Thus, it drastically influences the instrument complexity and sensitivity. Among the types of label molecules, fluorescent dyes are the most employed due to the high sensitivity of the fluorescence technique. In the following section we review the mechanisms associated with the first three probe categories above-reported being these the most suitable to be integrated into PoC systems. Intercalating Agents (First Type Probe). Numerous dsDNA intercalating agents have been proposed to detect real-time amplification. These compounds are featured by planar molecular structures able to intercalate into the two paired bases of dsDNA and interact with the minor groove.50 These molecules contains a transduction active center able to emit fluorescent or electrochemical signals (Figure 4a). Intercalative fluorescent labels are featured by the properties that once intercalated in the amplified product these molecules give a new emission occurring or band shift. This generates an increase in the fluorescent signal as the amplification proceeds (Figure 4b). The most popular and commercial fluorescent intercalating agents are ethidium bromide, SYBRGold, Evagreen and SYBRGreen. Several redox-intercalating probes have also been proposed in the literature for real-time PCR detection. They are organic molecules such as methylene blue,51 anthraquinone-pyrrole derivatives,52 and some metal complex based on osmium51,53 and ruthenium.54 These metal complexes are properly engineered with a redox active metallic center covalently bound to a planar intercalator ligand such as phenanthroline, bypiridine derivates, or dipyrido[3,2-a:20,30-c]phenazine (DPPZ). The redox intercalators are usually employed to read the signal at the electrode of the free intercalator molecule during the PCR cycles. In contrast with fluorescent labels, this strategy leads to redox signal decreasing during the PCR cycles (Figure 4c). In 2006 Chen et al. reported an interesting study on the multiple binding modes involved in the interaction of redox label probes based on Ru/Os-complexes with dsDNA.54 By means of square waves (SW) measurements they found two binding modes consisting of intercalation and electrostatic association. In 2011 Limonges published an article51 where a series of redox intercalative compounds were used as probes for electrochemical real-time PCR through SW measurements. It was demonstrated that the redox Os complex Os[(bpy)2DPPZ]+2 was the best probe since it strongly intercalated the amplified dsDNA products (Kb ∼ 5 × 106 M−1) giving a sensitivity of the method of about 103 copies/ reaction. This metal−organic complex was demonstrated to be chemically stable under PCR thermal cycling at the concentration optimized (0.5 μM) and not inhibit the amplification. E


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extension of circular target.65 Once denaturated, the ssDNA target is amplified by primers annealing and extension phases properly designed to occur at the same temperature. On this matter, there are extensive reviews in the literature.12 Therefore, we considered it useful only to report a summary (Table 2) of the main isothermal approaches developed so far. It can be noted that most of the isothermal methods are based on fluorescent detection. We found only two isothermal DNA amplification methodologies that can be coupled with an electrochemical detection, the helicase-dependent amplification (HDA)66 and the loop mediated isothermal amplification (LAMP).67 Isothermal PCR is very appealing for the integration in miniaturized PoC systems. In fact, it eliminates the need for accurate temperature cycling and control, reducing the cost of the battery-operated power and the instrument complexity. It is also versatile to be transduced by several detection methods including optical and electrical methodologies. Although isothermal PCR is the youngest PCR method, commercial products are already appearing in the market as discussed in the following section (i.e., Alerei product in Table 4). PCR Facilitator Agents. The PCR-inhibition effects are very relevant when clinical samples (such as saliva, blood, skin, sputum, swab, and so forth) are processed in a fully integrated PoC system. Indeed it is well-known that Taq enzyme is inhibited by cellular debris and components.85 To overcome these limitations several PCR-facilitators have been proposed. They typically are organic, inorganic, or biological species such as betaine, Triton X-100, PEG 400, Tween 20, glycerol, metal nanoparticles, and bovine serum albumin (BSA).86 The protection mechanism depends on the nature of the chemical agents. In the case of betaine (N,N,N-trimethylglycine), it has been found that it acts with two mechanisms: (i) destabilization of GC-rich DNA sequences; (ii) increasing of the thermal unfolding transition temperatures of proteins. This enhances both the yields and specificities of PCR amplification.86 The nonionic detergents (i.e., Tween 20 and Triton-X) are largely used as PCR facilitators because they stimulate the activity of Taq polymerase and enhance the amplification specificity.87 Glycerol facilitates the DNA amplification by several mechanisms including enhancing the hydrophobic interactions between protein domains, raising the thermal transition temperature of proteins and lowering the strand separation temperature of DNA.88 PEG polymers acts as PCR facilitator by affecting the thermal stability of the primers and the thermal activity profile of the Taq enzyme. They also possess enzyme stabilizing properties crucial to maintain enzymatic activity.89 BSA is the most common and low cost PCR-facilitator additive used to prevent the PCR inhibition in blood sample.90 Additionally, it is commonly used as passivation layer for microfluidic PCR-based systems. In our previous work we demonstrated that small amounts of BSA prevent PCR inhibition, allowing DNA amplification with a blood volumes larger than 100 pL.41 Metal nanoparticles have multiple effects: they stabilize the active Taq conformation91 by interacting with the DNA template increasing the efficiency of annealing;92 they enhance the thermal efficiency of the solution by heat dispersion.93 Table 3 summarizes the main advantages and disadvantages of the above-discussed PCR amplification chemistry and their suitability versus PoC integration.

Figure 5. Schemes of chemistry for (a) labeled hydrolysis probe and (b) labeled hybridization probe.

the distance between Q and R increases, inducing fluorescence emission due to FRET inhibition. This approach has the advantage of high specificity, with the probe sequences being designed for the specific target. The main limitations for the integration into PoC systems are related to the optical transduction method (not easy to be miniaturized) and the fluorophore stability that could limit the integration on the reagents on-board. Labeled Primer-Probe (Third Type Probe). An alternative approach combines the fluorescently labeled probe with the primer in a single oligonucleotide molecule (primer-probe). Depending on specific design of the primer-probe (Harpins,61 Cyclicons,62 Angler-probe63) and (Amplifluor prime-probe64), the fluorescence signal could be detected during the denaturation or the extension phase. As an example we report the case of the Amplifluor system, in which the signal is detected during primer extension upon the intramolecular hybridization of the incorporated labeled probes with the newly formed product, as schemed in Figure 6.

Figure 6. Schemes of chemistry for labeled primer-probe (Amplifluor system).

The labeled primer-probe approach includes both primer and probe sequence into the same labeled molecules, presents higher specificity with respect to the previously discussed methods. The main limitations are related to the length of the probe sequences that can provoke several drawbacks such us hairpin formation, increasing the hybridization temperature that can affect both PCR efficiency and power consumption. Also, in this case, the optical transduction method creates a limitation for the integration of miniaturized devices (vide supra). Isothermal PCR. To reduce the thermal complexity of realtime PCR, a new amplification method has been developed employing a single temperature to produce NA amplification (isothermal PCR). In this technology the ds denaturation is achieved by methods alternative to the thermal annealing at 95 °C, such as enzymatic duplex melting and primer annealing, F


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exponential amplification reaction (EXPAR) beacon-assisted detection amplification (BAD AMP)

strand displacement amplification (SDA) nicking enzyme amplification reaction/nicking enzyme mediated amplification isothermal chain amplification (ICA)

smart-amplification (Smart Amp)

cross-priming amplification (CPA)

In ICA method a single strand cutting is facilitated through RNase H and DNA−RNA−DNA chimeric primers, so that an intermediate target is generated using strand-displacing amplification via sacrificial outer bumper primers. This method was developed by RapleGene82 This method amplifies short trigger oligonucleotides, generated via “fingerprinting” reaction from adjacent nicking enzyme recognition sites in genomic DNA. The following rapid exponential amplification, mediated by a template sequence contains two copies of the trigger complement separated by the nicking enzyme recognition site83 In this method the NA are recognized by a molecular beacon. A DNA polymerase and nicking endonuclease are used to rapidly amplify the signal.84

In this approach a recombinase−primer complex scans the dsDNA facilitating the primer binding. After that a single strand binding protein furtherly stabilizes the denaturated strands. The rest of the reaction sequence is analogous to PCR72,73 The method is based on amplification of short DNA (or RNA) primer to form a long single stranded DNA (or RNA) by a circular DNA template and special DNA or RNA polymerases74 This methodology uses a properly designed circular probe in which the 3′ and 5′ ends are brought together in juxtaposition by hybridization to a target75 This method is based on a set of four properly designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the DNA target to start the LAMP reaction.76 This approach (developed by USTAR) is based on a strand displacement DNA polymerase without initial denaturation step. At the assay temperature of 63 °C, the formation of a primertemplate hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over reannealing of the template strands by the high concentration of primer relative to template DNA.77 Smart Amps was developed by Omics Science Center. This technology achieves its efficiency and accuracy by combining three key technologies: asymmetrical primer design, DNA polymerase with strand-displacement activity, and mismatch binding protein.78 This method developed by Becton Dickenson operates at a single temperature and makes use of a polymerase in conjunction with an endonuclease that will nick the polymerized strand so that the polymerase will displace the strand without digestion while generating a newly polymerized strand.79 The NEAR method developed by Ionian Technologies use nicking endonucleases that introduce a single strand cutting on only one strand80 In NEMA method an intermediate target is generated using strand-displacing amplification via sacrificial outer bumper primers. It was developed by USTAR81

principle of method

This method developed by Gen-Probe consists of RNA transcription-mediated amplification. It uses RNA transcription (RNA polymerase) and cDNA synthesis (reverse transcriptase) to produce RNA amplicon from a NA target.68 This method developed by BioMerieux is based on RNA transcription mediated by three enzyme (RT-DNA pol, RNase H, and RNA pol). The RNA target is converted to double strand cDNA, followed by RNase H degradation of the original strand and DNA polymerization initiated by a second primer69 The method uses two single-stranded oligonucleotide probes (extension and template). Thanks to a proper design, they can only anneal to each other in the presence of the specific target to form a three-way junction with target and extension probes to initiate linear RNA polymerization based amplification70 This method uses a DNA helicase to denaturate dsDNA for primer hybridization and subsequent extension. It was developed by Biohelix71

isothermal PCR

transcription mediated amplification (TMA) nucleic acid sequence-based amplification (NASBA) signal mediated amplification of RNA technology (SMART) helicase-dependent amplification (HDA) recombinase polymerase amplification (RPA) rolling circle amplification (RCA) ramification amplification (RAM) loop-mediated amplification (LAMP)

Table 2. Summary of the Main PCR Isothermal Approaches

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ACS Sensors Table 3. PCR Amplification Chemistry Comparison PCR technology RT-PCR

isothermal PCR

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chemistry intercalating agents

suitability versus PoC

advantages

disadvantages

high versatility (versus transduction methods and targets)

low specificity need of severe NA purification thermal cycling moderate complexity instrumentation (optical detection)

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fluorophore stability thermal cycling moderate complexity instrumentation (optical detection) fluorophore stability PCR efficiency thermal cycling moderate complexity instrumentation (optical detection) reagents cost

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labeled probes

active versus solid phase PCR low cost low complexity instrumentations (electrical detection) high specificity

labeled primer probes

very high specificity

various chemistry (seeTable2)

no thermal cycling easy to be integrated into PoC system low complexity instrumentation low power consumption

++

++++

reactors. In fascinating studies, Pipper and co-workers101,102 presented a “droplets-platform” able to detect the avian influenza virus H5N1 from throat swab in less than 28 min and 30 green-fluorescent protein (GFP) transfected in THP-1 cells from 25 mL of blood within 17 min (Figure 7a). This approach extremely simplifies the integration of microfluidic components, giving an excellent technological solution for inexpensive PoC chips. Therefore, the performances exhibited from the prototypes described earlier (vide supra) indicate that

GENETIC POC DEVICE SYSTEMS Genetic PoC is defined as a system able to perform in vitro genetic analysis by unskilled personnel near the patient, in hospital, in the physician office, clinic, or home with rapid answer and low cost94 as already discussed in the introduction section (see Figure2b). Therefore, based on the currently available biotechnologies for NA analysis (see previous section), these systems must be able to manage, integrate, and merge the fundamental steps required by molecular analysis (extraction, amplification, and detection) in a unique solution. This is, of course, a very challenging task since there are several basic sets of laboratory operations to be integrated and extensive multidisciplinary competences (chemistry, biochemistry, physic, microfluidic, molecular biology, engineering, software) to be synergistically harmonized. The main goals to be achieved are systems with low complexity and the integration of NA analytical methods sustaining performances in terms of sensitivity, specificity, and reliability at the level of the standard methods using commercial extraction and RTPCR kits. Therefore, while there is extensive literature reporting systems and miniaturized devices for PCR chip and some others on sample prep devices,11,95−97 there are only a limited number of studies where the whole process for NA analysis is a fully integrated solution in a PoC perspective. In this view, in the following sections we review the genetic PoC systems in two states of the development roadmap: literature studies representing fully integrated for PoC prototypes (fully integrated system prototypes), and finally, systems already in the market (PoC commercial products). Fully Integrated System Prototypes. In a fully integrated “sample-in answer-out” perspective, an innovative technology based on “droplet manipulation” has been developed during the past few years.98−100 The principle of this technology is the handling, through magnetic actuators (i.e., coils), of droplets having embedded superparamagnetic particles. These droplets contain reagents or the starting sample so that by merging and splitting, they are able to achieve all the fluidic functions and reactions required by NA analysis. Basically these droplets acts as virtual pumps, valves, mixer, SPE substrates, and RT-PCR

Figure 7. Fully integrated PoC systems prototypes: (a) droplets platform, reprinted with permission from ref 102. Copyright 2007 Nature Publishing Group. (b) electromagnetic droplets platform with plastic topographical barriers, reprinted with permission from ref 103. Copyright 2013 Elsevier. (c) Portable polycarbonate microfluidic system for bacteria detection, reprinted with permission from ref 104. Copyright 2010 Springer. (d) BioChipSet cassette, reprinted from ref 107 (free use from BioMedCentral). H


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greater than 500 bases. Additionally several tests versus real product have been performed, including reagent stability (at least 6 months at 22 °C) and robustness to shock and vibration. This system presents a maturity level very close to commercial products for military and forensic applications. Alongside these engineered fully integrated PoC prototypes, there are certain genetic PoCs products commercially available nowadays (see following section). Commercial Products. The commercial PoC platforms are listed in Table 4 together with their main features. The design of these systems reflects the target to be compact and provide fast, easy-to-use, on-demand testing. Basically in terms of system architectures all these platforms are composed of a disposable cartridge (mainly in plastic materials to contain the cost of the analysis) prefilled with all the necessary reagents, managed by an instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module, and analysis SW. Most of these employ extraction chemistry based on SPE using silicacoated magnetic beads (GeneXpert by Cepheid, Cobas Liat System by Roche, FilmArray by Biomerieux, ML by Enigma) or silica membrane (Idylla by Biocartis) coupled with fluorescent real-time PCR detection methodologies. A proprietary isothermal amplification technology, named NEAR (Nicking Enzyme Amplification Reaction), has been developed and implemented in the Alere-i platform with the advantage of delivering PCR results very fast in a few minutes (see Table 4). Note that all the commercial solutions described above employ fluorescence detection methods, in most of cases, integrated with real-time PCR amplification. This can be certainly attributed to the robust and consolidated reaction chemistry with affordable cost together with the fact that these methodologies prevent amplicon contamination reducing the risk of false positive results.14 Several diagnostic kits CE-IVD cleared are available for Strep A, influenza viruses, KRAS, respiratory panels, NRAS-BRAFEGFR492R, meningitis/encephalitis (ME) panel, blood culture identification (BCID) panel, gastrointestinal (GI) panel, and so forth (see Table 4). An interesting comparison study among some of the genetic PoCs reported earlier in the detection of Influenza A, B, and respiratory syncytial virus (SRV) has been published recently.108 It highlights the importance of studying the use of genetic PoC in a real near-patient setting, in addition to the clinical outcomes, also ease of use, patient and staff acceptability, and cost-effectiveness. These are the crucial aspects that can affect either the success or the failure of these portable technologies in clinical practice, allowing the extensive use of these molecular methods in decentralized environments. As can be noted from Table 4, despite the sample preparation and PCR technologies nowadays being very mature, relatively limited numbers of PoC products are currently on the market. This is because several challenges must be faced to develop a genetic PoC system. They include (i) the selection of reagents exhibiting extensive stability (up to 1 year) when they are on board; (ii) selection of sample preparation and PCR chemistry including few process steps; (iii) architectural integration of several system modules from thermal to microfluidic and optical parts, preserving the low cost, the ease of use and robustness of the device; (iv) selection of limited biocompatibility and low cost materials for the realization of the disposable PoC device. All these aspects make the development of genetic PoC for diagnostic use very

this has a very good potential for application in commercial products for infectious diseases diagnosis. Always in this field, Wang and co-workers103 proposed an electromagnetic droplets platform integrating NA extraction using silica-coated magnetic beads with RT-PCR on a single plastic cartridge (56 × 15 mm in size) (Figure 7b). The novelty of this approach is the automation of droplet processing using a method that combines the actuation by magnetic coils in tandem with by topographical barriers, to simply and efficiently control the droplet manipulation. The platform has been proven to detect in a sample-in answer-out format KRAS oncogene starting from 5 μL of human whole blood as the biological sample input. Based on both the exhibited performance (vide supra) and the simplification of the system architecture coming from magnetic droplet technology, this prototype system can certainly have potential in real applications in the field of genetic diseases. Bau and colleagues at University of Pennsylvania104,105 presented a portable fully integrated microfluidic system made of polycarbonate for bacteria detection (Figure 7c). The device consists of a plastic cassette (71 mm × 48 mm in size) integrating several fluidic components (blisters, valves, and chambers) for DNA extraction (employing SPE on silica membrane) and detection via RT-PCR (or CE, flow strip). The analyzer provides mechanical actuation for the on-chip pouches and valves for liquid pumping and flow control in the cassette. The analyzer also includes a pair of thermoelectric units to provide thermal cycling for RT-PCR. The device has been proven to be able to detect B. cereus Gram-positive bacteria spiked in 100 μL of saliva and viral armored RNA HIV and HIV I virus in saliva sample. In terms of its application in real products, this solution offers the advantages of low cost (plastic material) and good sensitivity to be used in the field of infectious diseases. Manage et al.106 developed an interesting low cost technology, named “gel strip”, consisting of a plastic chip filled with a special gel that embeds reagents for the analysis starting from unprocessed blood. Basically whole blood is added in the gel strip cassette (8.5 μL) and RT-PCR is directly run in the strip using a custom-constructed DNA polymerase that is not inhibited by whole blood. Detection of BK Virus (BKV) DNA was demonstrated up to 1.7 × 105 copies/mL. Even if this prototype does not exhibit very high sensitivity, it offers the relevant advantages of the simplicity of the system architecture and the low cost of the material used (plastic). Despite that this technology has been tested for viral applications, on the basis of both the exhibited sensitivity and the sample volume used, it could be a good candidate for real applications targeting human genomic DNA (genetic diseases). In a plastic format approach, Selden and colleagues107 reported a BioChipSet cassette able to perform sample-toanswer STR profile analysis starting from buccal swab (Figure 7d). The system, containing lyophilized reagents on board, integrates four injection-molded parts: (a) smart cartridge (the largest part 93 × 152 × 84 mm3) with reagents on board capable of purifying (using a silica membrane) five buccal swab samples in parallel; (b) the gel smart cartridge (33 × 56 × 28 mm3) containing the reagents for CE; (c) the integrated biochip (166 × 296 × 5.5 mm3) holding the PCR chambers; and finally (d) the S&D biochip (254 × 84 × 0.4 mm3) that analyzes the amplified sequence by CE. Results shown that, for each of 5 buccal swabs, the system is able to automatically detect 15 short tandem repeat loci and the amelogenin locus in 84 min. The device exhibited single-base resolution from 100 to I


J

Great Basin’s diagnostic system

Great Basin Scientific

Biomerieux

FilmArray

Biocartis

Idylla

Enigma

Roche

Cobas Liat System

ML

Cepheid

company

GeneXpert

name

Disposable. Cartridge containing (blister packs or lyophilized) all of the reagents required to run the test Instrument. Bench top instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW

Extraction. Magnetic beads based extraction

Detection. Integrated isothermal helicase-dependent amplification (HDA) and fluorescent detection

Detection. Nested multiplex PCR (PCR I to performs a single, large volume, multiplexed reaction; Individual, singleplex PCR II reactions to detect the products)

Extraction. Magnetic Beads

Detection. Real-Time PCR

Instrument. Bench top instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW

Instrument. Bench top instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW Disposable. Plastic cartridge prefilled with all the necessary reagents Instrument. Bench top instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW Disposable. Plastic cartridge with preloaded reagents


Extraction. Magnetic beads based extraction

Disposable. Plastic cartridge prefilled with all the necessary reagents

Extraction. Silica membrane


Intrument. Bench top instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW

Instrument. Bench top instrument integrating reagents, microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW Disposable. Plastic cartridge with preloaded reagents


Extraction. Chaotropic lysis and silica-coated magnetic beads

Disposable. On hand plastic cartridge integrating reagents

system architectures

Extraction. Integrated extraction based on magnetic beds

extraction/detection methods

Table 4. Genetic PoC Commercial Products

Respiratory Panel: (20 respiratory viruses and bacteria from Nasopharyngeal Swab) Meningitis/Encephalitis (ME) Panel cerebrospinal fluid (CSF) (14 most common pathogens Blood Culture Identification (BCID) Panel (24 pathogens and 3 antibiotic resistance genes) Gastrointestinal (GI) Panel tests for most common gastrointestinal pathogens Toxigenic Clostridium difficile (C. diff) Group B Streptococcus (GBS) Staph ID/R Blood Culture Panel Shiga Toxin Direct Test

Influenza A, B and respiratory syncytial viruses in respiratory specimens

20 min

Influenza A virus and Influenza B virus and respiratory syncytial virus RNA in nasopharyngeal swab specimens BRAF Mutation Test KRAS Respiratory Panel (IFV-SRV) NRAS-BRAF-EGFR492R

90−120 min

60 min

95 min

35−150 min

20 min

15 min

77 min

time to results

Influenza A virus and Influenza B virus RNA in nasopharyngeal swab specimens

Strep A (Streptococcus pyogenes (Group A β-hemolytic Streptococcus) in throat swab specimens)

MRSA NxG, SA Nasal Complete, MRSA/SA SSTI, MRSA/SA BC, C.difficile/Epi, vanA, Carba-R, Norovirus Flu, Flu/RSV XC, MTB/ RIF, EV, TV, CT/NG, GBS, GBS LB FII and FV

panels

CE IVD

FDA, CEIVD, and TGA certified

CE-IVD

RUOs

CE-IVD, 510 (k) cleared and CLIAwaived CE-IVD, 510 (k) cleared and CLIAwaived CE-IVD, 510 (k) cleared and CLIAwaived CE-marked IVDs

FDA approved CLIA moderate complexity

regulatory status ref

http://gbscience.com/ products/test-andpanels/

http://www. biomerieuxdiagnostics.com/ filmarrayr-multiplexpcr-system

http://www. enigmadiagnostics. com/

https://www.biocartis. com/idylla

https://molecular. roche.com/systems/ cobas-liat-system/

http://www.cepheid. com

ACS Sensors Review


Review http://www.genepocdiagnostics.com/ technology/

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NEW FRONTIERS AND PERSPECTIVES FOR POC The new frontiers in the genetic PoC are undoubtedly in the direction to continue the fully integration, compactness, and reduction of system complexity to integrate these systems in mobile smart devices such as phones and smart watches in Internet of Things (IoT) context.109 A first limitation in this direction concerns the most frequently used transduction method. As described in the previous section, most of the current integrated PoC systems employ fluorescent detection due to the good sensitivity, high specificity, real-time detection, and multiplying in the analysis. Additionally, it is easy to be incorporated into the reactions for NA detection.110 However, in terms of system architecture, this implies the integration of a series of optical components, such as excitation sources, photodetectors, and filters, external to the sensor device.111 Moreover, due to the accurate optical alignment required, optical transduction modules cannot be monolithically integrated with the active microchip where the NA reactions take place. This increases the cost of the final system and requires periodic calibration and maintenance. Therefore, this type of detection does not meet the criteria of portability mentioned above. Electrical detection methods (capacitive, resistive, or electrochemical transduction) have been proposed as a good alternative to the optical detection. In fact, they offer the advantages to be easily integrated into compact and smart devices, overcoming the limitations discussed above of compactness related to optical methods. Moreover, they are not mature technologies for NAs detection and, so far, lack multiplexing capability. Among the possible electrical detection methods, electrochemical detection is the most promising candidate to be used in PoC devices. In fact, it has the advantage of allowing high flexibility and real-time detection, although it exhibits sensitivity lower than the optical detection.51 Moreover, electrochemically active labels (such as metal-complex, organic molecules, and so forth) are typically more stable than the fluorescent dyes (Cy5, FAM, and so forth), being a very important aspect in their application in commercial products. A further limitation is the need for PCR amplification reactions that in terms of system structure require the presence of thermal modules including thermal components such as temperature sensors and heaters. This represents not only a drawback to reduce the system complexity, but also involves a certain energy consumption, making a limitation for the battery usage. A positive advancement for overcoming the thermal limitation has been done by the invention of the PCR isothermal methods (see PCR amplification chemistry section) and certainly year-to-date is recognized as the most suitable for PoC devices. Finally, the consolidated extraction protocols require several fluidic steps that in terms of system integration are translated into microfluidic functionalities requiring, depending on the technology employed for the actuation, several components such us valves, channels, and so forth. This again represents a limitation in the reduction of complexity of the final system. In the view of the above considerations and with the aim to overcome the current limitations, the new frontier of research for genetic PoC should address the following objectives: (i) Development of quantitative detection methods based on electronic transduction112,113 instead of optic transduction.

Clostridium difficile

70 min Influenza A and B Test GenePoC


Disposable. Plastic cartridge (named PIE) with preloaded reagents Instrument. Bench top instrument (Revogene) integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW

8 min Strep A

GenePoC

Extraction. Glass beads based extraction

13 min RSV Rapid Molecular Test

Instrument. Bench top instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module and analysis SW

ref

challenging to accomplish and so far they have limited the products available in the markets.

FDA CLIA Waiver FDA Clearance FDA Clearance CE Mark 15 min Influenza A and B Test Disposable. Plastic cartridge with preloaded reagents Extraction/Detection. Lysis/Isothermal Amplification Alere ALERE i

regulatory status time to results panels system architectures extraction/detection methods company name

Table 4. continued

http://www.alere-i. com/en/index/alerei-system/technology. html

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target hybridization (Figure 8b). In the absence of the target, the redox reporter gives a detectable current being very close to the surface (signal-on). Upon hybridization, the beacon probe is opened and the reporter is away from the surface with a consequent reduction of current signal (signal-off). The method has been integrated with PCR reaching a limit of detection (LOD) of ∼300 copies/50 μL of genomic DNA and, as already reported in the Towards Fully Integrated Systems, the detection of H1N1 virus with sensitivity 4 orders of magnitude lower than the clinical titers in typical throat swab samples.116 It was also coupled with LAMP amplification reaching a duplex electrochemical detection of both S. typhimurium and S. choleraesuis.115 Concerning the PCR-free methods, Wen et al.117 proposed the identification of MicroRNAs (miRNAs) up to attomolar concentration using a DNA nanostructure-based interfacial engineering approach (Figure 8c). This approach is based on a three-dimensional (3D) DNA tetrahedral nanostructure creating a sandwich structure with miRNA target and a signal probe labeled with biotin tag that, in turn, specifically binds to avidin-HRP which catalyzes the reduction of hydrogen peroxide and generates quantitative electrochemical current signals in the presence of the cosubstrate, 3,3′,5,5′-tetramethylbenzidine (TMB). Zhu et al.118 reported a strategy using two probes (capture probe covalently linked to magnetic beads (MB) and reporting probe (bar code) covalently linked to quantum dots (QD) hybridizing with miRNAs to form a three-component hybridization complex. The addition of ligase enzyme generates a covalently interlinked QD-MB-DNA forming a stable duplex with target miRNAs. The SW analysis of the joined QDs barcodes produced a sensitive electrochemical signal indicating the levels and pattern of the target miRNAs. We also recently proposed an electrochemical PCR-free method for the detection of pathogen genome (HBV virus).119 It has been achieved by a silicon miniaturized device containing three planar microelectrodes. The chemical strategy employed was based on a cooperative hybridization with two capture probes immobilized to the platinum working electrode surface and electrochemical detection using an intercalative agent ([Os(bpy)2DPPZ]Cl2). The system reaches a LoD comparable to the standard qRT-PCR method of 20 copies/reaction. These PCR-free methods, although offering a fascinating solution for future portable and easy-to-use products, are not mature technologies in comparison with the amplification methods. They have to overcome several challenges before being extensively used for NA detection. In fact, the general application for a wide range of genomes must be proved, together with extensive testing to ascertain the reproducibility and the robustness of the methods. In order to reduce the extraction and purification step, we recently proposed a sample preparation process in one step.41 The method uses a mix of proteases that lyse the human cells and digest most of the proteins present in the blood allowing the direct detection of betaglobin gene with PCR without further purification steps.

This will have enormous advantages to directly interface the device with the electronic circuitry having intelligence on board compactness. (ii) A second important goal of research will be the development of new biochemical strategies to allow NA detection without any amplification step (PCR-free methods). This, of course, will have a tremendous impact on the rapidity of the analysis as well as the reduction of the final system complexity and energy consumption. (iii) Finally, the development of innovative biochemical strategies to achieve extraction methods in very few steps, preferably one single step. These research objectives will enable great progress in the direction of the future perspective of ultracompact and smart devices described above. Several approaches have started from year to date from the many researchers to pursue these goals. In the fields of the NA electronic detection, an interesting electrochemical methodology has been developed by Limonges and co-workers at Université Paris Diderot using redoxintercalating probes (see RT-PCR Probe Chemistries section, Intercalative Agents, Figure 4c) during the PCR amplification. By implementing this detection methodology, they recently114 achieved a single DNA copy detection of bacteriophage M13mp18 employing real-time loop-mediated isothermal amplification (LAMP) and the redox reporter Os[(bpy)2DPPZ]+2 in only 30 min (Figure 8a).

Figure 8. (a) NA electrochemical detection by intercalative compound, reprinted with permission from ref 114. Copyright 2016 American Chemical Society. (b) PCR free method by DNA nanostructure-based probes, reprinted with permission from ref 115. Copyright 2015 American Chemical Society. (c) NA sequence-specific NA electrochemical detection reprinted with permission from ref 117. Copyright 2012 Nature Publishing Group. (d) PCR free method by cooperative hybridization and intercalative compound, reprinted with permission from ref 119. Copyright 2017 Royal Society of Chemistry.

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CONCLUSIONS In this review we have described the most recent advances in the genetic PoC technologies. Genetic PoC are systems integrating in a portable “sample-in-answer-out” format all steps necessary for the molecular analysis, sample preparation (extraction and purification of NA), and detection (i.e., PCR or

Soh and co-workers at University of California developed a smart strategy for sequence-specific NA electrochemical detection.115 It employs a redox-reporter-modified molecular beacon probes (see RT-PCR Probe Chemistries section, Labeled Probes; Figure 5b), covalently linked to the electrode surface. This approach gives an on−off current signal upon L


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(10) Saiki, R. K.; Scharf, S.; Faloona, F.; Mullis, K. B.; Horn, G. T.; Erlich, H. A.; Arnheim, N. Enzymatic amplification of beta-globin Genomics sequences and restrictyion site for diagnosis of sickle cell anemia. Science 1985, 230, 1350−1354. (11) Gill, P.; Ghaemi, A. Nucleic Acid Isothermal Amplification TechnologiesA Review, Nucleosides. Nucleosides, Nucleotides Nucleic Acids 2008, 27, 224−243. (12) Almassian, D. R.; Cockrell, L. M.; Nelson, W. M. Portable nucleic acid thermocyclers. Chem. Soc. Rev. 2013, 42, 8769−8798. (13) Higuchi, R.; Dollinger, G.; Walsh, P. S.; Griffith, R. Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 1992, 10, 413−417. (14) Wolffs, P.; Norling, B.; Rådström, P. Risk assessment of falsepositive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells. J. Microbiol. Methods 2005, 60, 315− 323. (15) Gubala, V.; Harris, L. F.; Ricco, A. J.; Tan, M. X.; Williams, D. E. Point of Care Diagnostics: Status and Future. Anal. Chem. 2012, 84, 487−515. (16) Niemz, A.; Ferguson, T. M.; Boyle, D. S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 2011, 29, 240− 250. (17) Nichols, J. H. Clin Point of Care TestingLab Med. 2007, 27, 893− 908. (18) Meyer-Rath, G.; Schnippel, K.; Long, L.; MacLeod, W.; Sanne, I.; Stevens, W.; Pillay, S.; Pillay, Y.; Rosen, S. The Impact and Cost of Scaling up GeneXpert MTB/RIF in South Africa. PLoS One 2012, 7, e36966. (19) Jones, G.; Steketee, R. W.; Black, R. E.; Bhutta, Z. A.; Morris, S. S. How many child deaths can we prevent this year? Lancet 2003, 362, 65−71. (20) Mabey, D.; Peeling, R. W.; Ustianowski, A.; Perkins, M. D. Tropical infectious diseases: Diagnostics for the developing world. Nat. Rev. Microbiol. 2004, 2, 231−240. (21) Grand View Research; http://www.grandviewresearch.com/ industry-analysis/molecular-diagnostics-market. (22) QIAamp DNA Mini and Blood Mini Handbook instruction for use QIAGEN; 2016; https://www.qiagen.com/it/. (23) MagaZorb DNA Mini-Prep Kit Technical Bulletin, Instructions for Use of Product(s), MB1004; 2011; https://www.promega.com. (24) Sortino, S.; Petralia, S.; Condorelli, G. G.; Conoci, S.; Condorelli, G. Novel Photoactive Self-Assembled Monolayer for Immobilization and Cleavage of DNA. Langmuir 2003, 19, 536−539. (25) Bimboim, H. C.; Doly, J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979, 7, 1513−1523. (26) Miller, S. A.; Dykes, D. D.; Polesky, H. F. A simple salting out procedure for extracting DNA from human cells. Nucleic Acids Res. 1988, 16, 1215−1219. (27) Pang, Z.; Al-Mahrouki, A.; Berezovski, M.; Krylov, S. N. Selection of surfactants for cell lysis in chemical cytometry to study protein-DNA interactions. Electrophoresis 2006, 27, 1489−1494. (28) Yuan, S.; Cohen, D. B.; Ravel, J.; Abdo, Z.; Forney, L. J. Evaluation of methods for the extraction and purification of DNA from the human microbiome. PLoS One 2012, 7, e33865. (29) http://www.zygem.com. (30) http://www.viagenbiotech.com. (31) Tongeren, S. P.; Degener, J. E.; Harmsen, H. J. M. Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR. Eur. J. Clin. Microbiol. Infect. Dis. 2011, 30, 1053−1061. (32) Gross, V.; Carlson, G.; Kwan, A. T.; Smejkal, G.; Freeman, E.; Ivanov, A. R.; Lazarev, A. Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies. J. Biomol. Technol. 2008, 19, 189−199. (33) Halstead, F. D.; Lee, A. V.; Couto-Parada, X.; Polley, S. D.; Ling, C.; Jenkins, C.; Chalmers, R. M.; Elwin, K.; Gray, J. J.; Gomara, M. I.; Wain, J.; Clark, D. A.; Bolton, F. J.; Manuel, R. J. Universal extraction

real-time PCR and isothermal methods). The use of these technologies enables fast and low cost in vitro genetic analysis by unskilled personnel near the patient, in decentralized environments such as hospital, the physician’s office, clinic, or home. We have identified two states of the development: literature studies representing fully integrated PoC prototypes (fully integrated system prototypes) and systems already on the market (PoC commercial products). The architectures of all these platforms are composed of disposable cartridges (mainly in plastic materials to contains the cost of the analysis) prefilled with all the necessary reagents, managed by an instrument integrating microfluidic actuation, thermal actuation module, optical fluorescence detection module, and SW analysis. The new frontiers in the genetic PoC are undoubtedly in the direction to continue full integration, compactness, and reduction of system complexity to create ultracompact devices that are smarter and faster with analytical performance superior to that of the current state-of-the-art. The most futuristic vision is to have these systems fully integrated in mobile devices such as phones and smart watches in Internet of Things (IoT) context. Genetic PoC will open the perspective of wide use of the molecular methods in the medical practice with a great impact on the prevention and early diagnosis of diseases.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Sabrina Conoci: 0000-0002-5874-7284 Author Contributions

Both authors contributed equally. Notes

The authors declare no competing financial interest.

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REFERENCES

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