Fortifying the diagnostic-frontiers with nanoscale technology amidst the COVID-19 catastrophe

KEYWORDS:

• COVID-19
• nanodiagnostics
• nanosensor
• nucleic acid
• SARS-CoV-2

The power of the small has been realized time and again in human history. The recent global fiasco, COVID-19, interlaced with unprecedented biomedical consequences and human loss, modifications in nutritional habits and physical activity, as well as tremendous impacts on economic, political, social, and psychophysiological health status [1,2], ushered in by the ‘petite yet powerful’ and ‘biochemically and genetically guileful’ SARS-CoV-2 [3] testifies the veracity of the statement. Amidst the global race to discover antiviral agents, neutralizing antibodies, and safe and effective vaccines [3,4], the denigration faced by governments of different countries for the deplorable mass testing efficiency, scarcity of sufficient testing kits as well as the asymptomatic viral transmission (manifested as the Achilles’ heel amidst the containment strategies) project the expansion and upscaling of prompt, sensitive, and effective SARS-CoV-2 testing strategies, executed in a well-timed, tactical, and regular mode as a focal global requisite. The current detection approaches for COVID-19 relies on clinical characteristics, epidemiological history, chest imaging, and laboratory diagnosis [5]. Thus, this write up aims to present a highlight of the current endeavors and progress in harnessing nanoscale-based diagnostic technology vis-à-vis the various pros and cons of the generic approaches for COVID-19 detection.

At this juncture, one may easily perceive the fact that a clear understanding of the genomic and the proteomic makeup of the pathogen as well as the alterations in the proteins/genes’ expression pattern in the host during and post-infection is a pre-requisite for the development of advanced diagnostic platforms including nano-based techniques. Similar to other coronaviruses, SARS-CoV-2 possesses a positive-sense single-stranded genomic RNA (with a 5′-cap and a 3′-poly(A) tail), approximately 30 kb in length [6]. Transcriptional regulatory sequences (TRSs) precede the 14 open reading frames (ORFs) in its genome; at the 3′ end, four structural proteins (spike, envelope, membrane, nucleocapsid) are encoded while nine putative ORFs for accessory factors intersperse the structural genes [6]. On a note of pertinence, the invitation to the virus into human cells is placed by the human angiotensin-converting enzyme 2 (ACE2) (expressed in a plethora of cells and tissues) that interacts with the receptor-binding domains (RBD) of the spike (S) glycoprotein [4,6]. In fact, coronaviruses represent one of the few RNA viruses, endowed with genomic proofreading potency and a deadly dynamism, marked by frequent swapping of RNA chunks between distant coronavirus relatives, resulting in intimidating versions or mutants with the capacity to infect new cell types and jump across species [7]. The molecular backdrop of the host–pathogen interaction, the immunological aspects, and the pathophysiology of the disease are gradually unfolding, besides various biomarkers, associated with COVID-19 have been identified [8], thereby, opening avenues for expanding the diagnostic frontiers.

Having said that, the initial screening and identification of SARS-CoV-2, the viral etiology of COVID-19 relied on the concert of computed tomography-based imaging/detection of the radiological changes in the lungs (within 1–2 h, though marked with low sensitivity), whole genome sequencing and electron microscopic visualization of the virus particles. The high cost, the requirement of high viral burden, expertise in viral recognition and the labor-intensive nature of the latter needs no elaboration. On the other hand, viral culture aids in the isolation of many viruses for subsequent research, however, prolonged time consumption (2–3 days), biosafety concerns as well as the requirement of expertise for maintenance of cell cultures and interpretation of characteristic cytopathic effects are the major challenges. Amongst others, the molecular detection techniques [5] for SARS-CoV-2 encompass RT-PCR and different nucleic acid-based techniques including nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP). Albeit, the selectivity and sensitivity of the conventional real-time RT-PCR are well established, howbeit, the requisite of trained personnel, accessibility of large and expensive instruments and reagents in specific laboratory settings, and time-intensive attribute pose serious feasibility constraints in environments with limited resources. Failure to identify recovered patients with past infection, chances of failed amplification due to inhibitors, and the possibility of false-negative results due to genetic variability are challenges for both conventional (turn-around-time, TAT: 3–4 hours) and sample-to-answer type real-time RT-PCR (TAT < 1 h) approaches. Techniques such as nanopore target sequencing (NTS) (based on the concert of target amplification and long-read, real-time nanopore-sequencing) [9] have also been exploited recently. On the other side of the coin, the applicability of serological and immunological tests, although, yield prompt results (< 1 h) with minimal equipment is mired in the context of the fact that the symptoms-onset in a patient for measurable antibody response may require several days to weeks. Furthermore, antibody production may fail in immunocompromised hosts while the possibility of cross-reactivity among similar viruses and subjective interpretation is projected as major limitation. In this milieu, one may easily comprehend the indispensability of inexpensive, easy to operate and sensitive diagnostic strategy offering a prompt and uninterrupted assessment of SARS-CoV-2 to facilitate better management of the disease and offer assistance to physicians in the treatment of COVID-19-related conditions. Pertinently, endeavors to develop CRISPR-Cas technology [10] based detection as well as point-of-care (POC)/bedside testing technologies such as lateral flow assays (LFAs) [5] merit special mention.

Amongst others, harnessing nanomaterials and the unique characteristics (attributable primarily to their high surface area to volume (S/V) ratio and quantum effects) at the nanoscale for the effective detection/diagnosis of the virus has fetched considerable research thrust. Application of various nanomaterials (such as graphene sheets, gold nanoparticles and magnetic field responsive nanoparticles, etc.), produced via inexpensive and facile protocols has opened up new portals for the fabrication of nanobiosensors with high precision, great sensitivity, and short response time for molecular detection of various biomarkers associated with the disease. The use of extremely low volumes of reagents and chemicals as well as the miniaturized size of the field-deployable nanoscale based platforms, are obvious advantages over the conventional approaches, particularly under constrained resource availability. Short TAT, on the other hand, has another apparent advantage in terms of decreasing the duration of waiting for a patient to get the results, consequently, reducing anxiety and augmenting compliance. Nanobiosensors have not only facilitated the expansion of non-polymerase chain reaction diagnostic technologies, but have also enabled selective naked-eye detection of the viral RNA (illustrated in the subsequent section) and easy to operate, low-cost nanobiosensor-based POC diagnosis (as in biochip assay formats) besides holding potential for multiplexing, integration with therapeutics and advancement of personalized medicine. The most important aspect, dictating the performance of the nanomaterials in achieving precise detection of the target-entity (e.g. SARS-CoV-2 target sequences in a multigene mixture) and analytical quantification of biochemical information is the design of the biosensing interface. On a specific note, nanomaterials, functionalized with antibodies or nucleic acids embody the prime entities for nano-based diagnostic technologies (Figure 1 [I]) based on antigen-binding or colorimetric assays, exploiting optical and photothermal platforms [11].

Figure 1. [I] Nanotechnology-based sensors for SARS-CoV-2 detection, involved in the development of platforms for viral tagging and nano-diagnostic assays (Abbreviations: Ab, antibody; FRET, Förster resonance energy transfer; LSPR, localized surface plasmon resonance; NPs, nanoparticles; PNA, peptide nucleic acid; PPT, photothermal therapy). (Reproduced with permission from Talebian et al. (2020) Copyright © 2020, Springer Nature Limited)

By the same token, exploiting the blueprint of life-DNA (quintessentially a bionanomaterial), for the fabrication of higher-ordered nanomaterials, facilitating the detection of the virus is an attention-grabbing endeavor. In this regard, self-assembly of long DNA strands and self-quenching probes (H1) was adroitly harnessed for the fabrication of DNA nanoscaffolds [12] (Figure 1 [II]). Hybridization of free H2 DNA probes and H1, mediated in the presence of SARS-CoV-2 RNA instantly resulted in an illuminated DNA nanostring. Pertinently, with a short reaction time of 10 min, facilitated by the localized acceleration of the DNA probes and a high signal gain documented within mild conditions (15–35 °C), the reported DNA nanoscaffold hybrid chain reaction (DNHCR) strategy was validated for saliva and serum samples. However, the requisite of equipment-based assistance for output of fluorescent signal in the reported protocol could be projected as a practical snag in meeting POC and user-friendly self-test requirements.

On a note of pertinence, one may easily perceive the relevance of effective sample collection protocols, circumventing any negative trade-offs with the biosafety measures. However, precise assays also demand efficient and automated nucleic acid extraction from the samples to skirt off false-negative outcomes or cross-infection. In this regard, a one-step lysis-binding combinatorial protocol for magnetic field mediated viral RNA-purification with the feasibility of direct introduction of poly(amino ester) with carboxyl groups (PC)-coated superparamagnetic nanoparticles (pcMNPs)-viral RNA complexes into subsequent RT-PCR reactions has been adeptly harnessed to significantly reduce turnaround time and operational requisites for molecular diagnosis of COVID-19 [13]. Based on the identification of ORFlab and N gene of the viral RNA, a10-copy sensitivity and linear correlation between 10 and 10⁵ copies of SARS-CoV-2 pseudovirus particles were documented. Extrapolating this, magnetic nanomaterials with virus-specific receptors-coating could find application in magnetic extraction/purification of the viral particles.

On the other hand, an important feature of the viral infection is the emission of volatile organic components (VOCs) (that may serve as disease-specific biomarkers) by the viral agents and/or their microenvironment [14]. The emergence of VOCs in exhaled breath, particularly in the initial stages of the infection could be exploited for the prompt detection of COVID-19. In this context, a breath device, comprising a nanoscale hybrid array of cross-reactive, chemically diverse chemiresistors of stabilized spherical gold nanoparticles (GNPs; core diameter: 3–4 nm) based semi-selective sensory units, mimicking the sensing strategy in natural mammalian olfactory systems and endowed with multiplexed detection efficacy, has been employed among a clinical study cohort of 49 confirmed COVID-19 patients (sampled twice: during the active diseased-state and post recovery), 58 healthy controls besides 33 non-COVID lung infection controls in Wuhan, China [15] (Figure 1 [III]). Pertinently, the accuracy of (94% and 76%) and (90% and 95%) were recorded post analysis of the training and test set data, respectively, in discriminating patients from controls and COVID-19 positive patients from patients with non-COVID lung infections. These arrays could be adapted to detect a wide gamut of biochemical signatures and for varied applications based on pattern discerning and machine learning algorithms.

Amongst other, a recent work had yielded graphene nanoplatelets coated paper-based low-cost electrochemical sensor chip with gold nanoparticles, decorated with N gene-specific antisense oligonucleotides (ss-DNA) for digital detection of SARS-CoV-2 nucleic acid [16] (Figure 1 [IV]). Besides, differentiating the positive COVID-19 clinical samples and the negative ones with absolute precision, the practicability of the sensor during likely events of viral genomic mutation has been projected to hold valid in the context of the unique fabrication protocol of the ssDNA-coated nanoparticles which concomitantly targets two distinct regions of the N gene. The distinctive features of the sensor were (a) significant augmentation of the output signal only in the presence of SARS-CoV-2 RNA, within less than 5 min of the incubation period, (b) limit of detection (LOD) of 6.9 copies/μL, circumventing the requisite of additional amplification, and (c) sensitivity of 231 (copies μL^–1)⁻¹.

Research has also been directed to adroitly harness gold nanoparticles, decorated with thiol-modified antisense oligonucleotides (ASOs), specific for SARS-CoV-2 N-gene for ‘naked-eye’ detection of positive COVID-19 cases within 10 min [17]. The colorimetric assay relied on (a) the selective aggregation of the nanoparticles, (b) modulation in the surface plasmon resonance in the presence of target RNA sequences, and (c) cleavage of RNA strand from RNA-DNA hybrid post addition of RNase H and scaled up nanoparticles’ agglomeration, yielding visually traceable precipitate in the solution (Figure 1 [V] A and B). In a similar vein, researchers from Switzerland had fabricated a dual-functional localized surface plasmon resonance (LSPR) biosensor via coalescing photothermal effect and plasmonic sensing transduction for the detection of SARS-CoV-2 viral nucleic acid [18]. The plasmonic chip with 2D gold nanoislands (AuNIs) was endowed with the potency to generate local plasmonic photothermal (PPT) heat and mediate the in situ hybridization for sensitive (LOD: 0.22 ppm) and precise detection of SARS-CoV-2 target sequences in a multigene mixture. Successful detection of SARS-CoV-2 in culture medium (LOD: 1.6 × 10¹ PFU/mL) and clinical (nasopharyngeal swab) samples (LOD: 2.42 × 10² copies/mL) besides non-quantifiable cross-reactivity with MERS-CoV antigen had attested the efficacy and high responsiveness of a SARS-CoV-2 spike antibody conjugated graphene sheets based field-effect transistor (FET)-biosensing device [19]. The use of mouse anti-human IgG antibody, labeled with lanthanide-doped polysterene nanoparticles (LNPs) as a fluorescent reporter-based lateral flow immunoassay (LFIA), relying on recombinant nucleocapsid phosphoprotein, dispended onto nitrocellulose (NC) membrane is another innovative, rapid (~10 min detection-period) and sensitive strategy [20]. This research-report from China attested the applicability of the immunodiagnostic method for positive identification of clinically suspicious cases of COVID-19, besides holding the potential to assess the disease progression and patients’ response to treatments.

High false-negative rates, protracted incubation periods and low sensitivity, associated with the current recommended molecular diagnostic protocols based on nucleic acid detection, as well as their failure to discriminate other respiratory virus infections result in patient misdiagnosis, encumber the containment strategies and hinder the timely response of the public, complicated additionally by the asymptomatic spread of COVID-19 as previously stated. The return to ‘normalcy’ undoubtedly is dependent on the wide-scale access to prompt, inexpensive and sensitive tests to diagnose actively infected individuals. High sensitivity and desirable LoD [16,18], high accuracy [17], low TAT (<15 min) [12,17,20], high specificity, avoidance of chances of cross-infection and false-negative outcomes [13,19], feasibility of integration into various platforms like breath device, paper-based electrochemical sensor chip, LFIA, etc., [16,17,20] high signal detection within mild conditions [12], requirement for lower volumes of reagents, low cost, portability, and possibility of even ‘naked eye’ detection [17] seem to confer special niche to nanomaterials in the realm of diagnostic technology for COVID-19. Integration of the nanobiosensors to smartphones via Wi-Fi/Bluetooth or with micro-controllers and LED screens is envisaged to open up new portals at POC settings. The feasibility of using nanosensors, incorporated into portable devices to detect active infections from crude biofluids would aid in rapid testing. Future research innovation may be streamlined toward developing a standard protocol/toolkit for developing nanosensors against a wider gamut of viral targets, without compromising with the detection potential in case of considerable mutation in the nucleic acid of the SARS-CoV-2 and linked biomarkers. An optimized concert of nano-enabled viral biosensing, Internet of Things (IoT), and artificial intelligence (AI) could pave ways for error-free, smarter management at a personalized level. In conclusion, various nanomaterials, with their unique tailorable physicochemical attributes, amenability for diverse biofunctionalization, quantifiable interactions at the biointerface through diverse modes, as well as the feasibility for integration into various platforms, could be the game-changer in the market as far as the development of low cost and high sensitive COVID-19 detection technology, yielding robust, rapid, and reproducible results, is concerned. Nanoscale technology, besides transcending the COVID-19 era, would be instrumental in offering neoteric diagnostic platforms and therapeutic approaches to dodge and deal with plausible future global health challenges.

Expert Opinion

The detection of SARS-CoV-2 relies on clinical characteristics, epidemiological history, chest imaging, and various laboratory diagnosis protocols, marked with respective pros and cons. Nanomaterials, known for their remarkable, tailorable and diverse physicochemical characteristics and stability, complemented by the availability of a plethora of biofunctionalization-strategies as well as diverse modes for gauging interactions at the biointerface. Besides, the practicability of integration into various platforms including POC devices, could be the game-changer as far as the development of rapid, robust, low cost, and high sensitive COVID-19 detection technology is concerned.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.