Researchers at Stanford have developed a genetic microlab that can detect COVID-19 in just a half-hour

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Throughout the pandemic, infectious disease experts and frontline medical workers have asked for a faster, cheaper and more reliable COVID-19 test.

Now, leveraging the so-called “lab on a chip” technology and the cutting-edge genetic editing technique known as CRISPR, researchers at Stanford have created a highly automated device that can identify the presence of the novel coronavirus in just a half-hour.

“The microlab is a microfluidic chip just half the size of a credit card containing a complex network of channels smaller than the width of a human hair,” said the study’s senior author, Juan G. Santiago, the Charles Lee Powell Foundation Professor of mechanical engineering at Stanford and an expert in microfluidics, a field devoted to controlling fluids and molecules at the microscale using chips.

The new COVID-19 test is detailed in a study published on Nov. 4 in the journal Proceedings of the National Academy of Sciences. “Our test can identify an active infection relatively quickly and cheaply.

It’s also not reliant on antibodies like many tests, which only indicates if someone has had the disease, and not whether they are currently infected and therefore contagious,” explained Ashwin Ramachandran, a Stanford graduate student and the study’s first author.

The microlab test takes advantage of the fact that coronaviruses like SARS-COV-2, the virus that causes COVID-19, leaves behind tiny genetic fingerprints wherever they go in the form of strands of RNA, the genetic precursor of DNA.

If the coronavirus’s RNA is present in a swab sample, the person from whom the sample was taken is infected.

To initiate a test, liquid from a nasal swab sample is dropped into the microlab, which uses electric fields to extract and purify any nucleic acids like RNA that it might contain.

The purified RNA is then converted into DNA and then replicated many times over using a technique known as isothermal amplification.

Next, the team used an enzyme called CRISPR-Cas12 – a sibling of the CRISPR-Cas9 enzyme associated with this year’s Nobel Prize in Chemistry—to determine if any of the amplified DNA came from the coronavirus.

If so, the activated enzyme triggers fluorescent probes that cause the sample to glow. Here also, electric fields play a crucial role by helping concentrate all of the important ingredients – the target DNA, the CRISPR enzyme and the fluorescent probes – together into a tiny space smaller than the width of a human hair, dramatically increasing the chances they will interact.

“Our chip is unique in that it uses electric fields to both purify nucleic acids from the sample and to speed up chemical reactions that let us know they are present,” Santiago said.

The team created its device on a shoestring budget of about $5,000. For now, the DNA amplification step must be performed outside of the chip, but Santiago expects that within months his lab will integrate all the steps into a single chip.

Several human-scale diagnostic tests use similar gene amplification and enzyme techniques, but they are slower and more expensive than the new test, which provides results in just 30 minutes. Other tests can require more manual steps and can take several hours.

The researchers say their approach is not specific to COVID-19 and could be adapted to detect the presence of other harmful microbes, such as E. coli in food or water samples, or tuberculosis and other diseases in the blood.

“If we want to look for a different disease, we simply design the appropriate nucleic acid sequence on a computer and send it over email to a commercial maker of synthetic RNA. They mail back a vial with the molecule that completely reconfigures our assay for a new disease,” Ramachandran said.

The researchers are working with the Ford Motor Company to further integrate the steps and develop their prototype into a marketable product.


Detection methods
Early detections of SARS-CoV-2 are very useful in the initial diagnosis and this will be quite valuable in controlling the spread of the infection. Hence, a significant approach towards rapid and accurate detection of virus is necessary. Several methods used for the detection of SARS-CoV-2 are the most commonly used ones that are either molecular tests or serological tests. The viral or molecular tests often indicate the active infection and these are designed to detect the genetic material of the virus. On the other hand, serological tests can detect the antibodies present in the blood and tissues produced during the fight against the virus, but these tests do not show about the current infected state.

Recent developments in molecular biotechnology have facilitated nucleic acid detection methods that are growing rapidly as the revolutionary technology, since polymerase chain reaction-based methods are advantageous to provide high sensitivity and rapid detection. Further, non-PCR-based methods are developed for the detection of SARS-CoV-2 RNA, which involves nucleic acid sequence-based amplification and isothermal nucleic acid amplification.

Polymerase chain reaction-based methods
PCR is a vital tool used in molecular biology to make millions to billions of DNA copies rapidly. It is very much advantageous to the medical fraternity that uses a small sample of DNA and amplifies it to significant amount for detailed investigations. This process involves initially separating a DNA strand containing the gene segment and a primer can be used to mark its location. Further, DNA polymerase accumulates a copy to each separated strand and then copies the copy continuously. The advantages and wide range of applications of PCR–based technique can be routinely and reliably used for detecting SARS-CoV-2 [25], [26]. Since SARS-CoV-2 consists of RNA as a genetic material, a reverse transcription is also carried before the PCR followed by product determination using appropriate detection methods or an instrumental analysis, which includes sequencing or gel visualization [27], [28]. Diagnosis in the early infection stage is more helpful and hence, real-time reverse transcriptase-PCR is predominantly used than the conventional PCR assay [29].

Van Elden et al. [30] described some disadvantages of RT-PCR methods such as contamination, time consumption, sample handling and analysis of post PCR can be easily avoided using TaqMan-based real-time RT-PCR. The sensitivity of this method was further improved by Yip et al. [31] using two TaqMan probes as a replacement for one probe for the detection of SARS-CoV. However, massive efforts are needed to overcome the difficulties in clinical detection such as lack of safe and external positive controls (EPC). EPC is an important component, which when the problem was avoided by using the armoured RNA as EPC for the detection of SARS-CoV [32] having the low detection limit of 10 copies /µL. Further, significant consideration should be given to reduce the risk of false negative results due to the variation in genome sequence due to genetic diversion caused as a result of rapid mutation in corona viruses. In such cases, multiplex RT-PCR is favourable to detect via multi-targeting detection of the coronavirus. Distinguishing between the non-pathogenic and pathogenic strains by using the mismatch-tolerant molecular beacons [33] demonstrated the detection limit of 5 copies/reaction [33].

Non-polymerase chain reaction-based methods
PCR-based techniques are widely used, but these techniques require the separation of DNA strands using a thermocycling, which limits its application in actual field applications. Isothermal nucleic acid amplification-based (INAA) methods developed in the past two decades without using thermocycler machine are useful for the detection of nucleic acid target sequence.

Loop-Mediated Isothermal Amplification (LAMP) is one of the INAA method with higher efficiency commonly used for the amplification of DNAs and RNAs exhibiting higher sensitivity and specificity. This method involves the use of a DNA polymerase along with four sets of specially designed primers that identifies six distinct sequences on the targeted DNA [34]. Gel electrophoresis is another commonly used approach to analyse the amplified products after LAMP essay. Poon et al. [35] and Pyrc et al. [36] demonstrated a LAMP reaction for SARS-CoV with the detection rates similar to those of the conventional PCR-based methods and 1 copy of RNA template per reaction, respectively.

The problem of virus detection can be simplified by using the precipitation of magnesium pyrophosphate or fluorescence dyes monitoring turbidity [37]. Shirato et al. [38] demonstrated one such a procedure for the detection of MERS-CoV RNA with the capability of detecting 3.4 copies without cross reaction with other respiratory viruses. Further, Thai et al. [39] demonstrated photometric method of measuring turbidity in one-step single-tube accelerated real-time quantitative for SARS-CoV having the sensitivity of 100-folds more than that of the conventional RT-PCR with a detection limit of 0.01 plaque formation unit.

In the above mentioned methods, the problems aroused due to the fact that primer dimer or non-primer reactions cannot be excluded as these techniques rely on nonspecific signal transducers such as solution turbidity or fluorescence dye intercalation. In such situations, sequence-specific LAMP-based methods that can readily separate nonspecific noise with true signal may be advantageous. In this direction, Huang et al. [40] proposed a method using RT-LAMP-VF (RT-LAMP and a vertical flow visualization strip) for the detection of MERS-CoV with a detection limit of 10 copies/µL. However, the major contributions from Ellington’s group improved in terms of specificity, reliability and LAMP detections. Replacement of dye in fluorescence detection with toehold-mediated strand exchange reaction for RT-LAMP-VF showed the detection of 0.02 to 0.2 PFU MERS-CoV without cross reaction with other respiratory viruses [41]. Du et al. [42] demonstrated a method combining strand exchange signal transduction, LAMP and a glucometer for the detection of MERS-CoV with a sensitivity of 20–100 copies/μl, equating to atto-molar.

Rolling circle amplification (RCA) is another technique that has attracted various scientific groups in nucleic acid determination. It is a unidirectional process of replicating nucleic acid producing numerous copies of circular molecules of DNA and RNA capable of 109 folds of amplification of each circle in 90 min under isothermal conditions. Further, the use of RCA has advantages such as requirement of minimum reagents and exclusion of false-positive results, which are frequently observed in PCR-based methods. An efficient method for sensitive detection of SARS-CoV genome using RCA in both liquid and solid phases was proposed by Wang et al. [43].

Microarray based methods
A typical microarray experiment involves the hybridization of an mRNA molecule to DNA template from which it is originated. Many DNA samples are used here to construct an array. The amount of mRNA bound to each site on the array indicates the expression level of various genes and this number may run in thousands. All the data are collected and a profile is generated for gene expression in the cell.

Shi et al. [44] designed 30 specific 60 mer oligonucleotide microarray based on TOR2 sequence in clinical samples that was successfully used for detecting SARS coronavirus. These designed microarrays represent the whole genome of SARS coronavirus, which was printed into an oligo microarray, and then applied to hybridizing with the samples of SARS patients treated and labelled by RD-PCR. This method offered results for seven samples hybridized on the microarray with no signals on blank and negative probe sites. Rapid mutation in SARS-CoV associated with 27 single nucleotide polymorphism mutations among the spike gene has lead to epidemicity. Considering such a mutation problem in SARS-CoV, Guo et al. [45] designed a microarray based on a single nucleotide polymorphism DNA, which would detect and genotype these single nucleotide polymorphisms and allow us to understand the pathogenicity and epidemicity of a given strain. Amplified products of cDNA from PCR technique of different strains of SARS-CoV were hybridized on the fabricated microarray. This method detected 24 single nucleotide polymorphism and the method was helpful to identify the strain with 100% accuracy for 19 samples in detecting and genotyping.

The sudden outbreak of viruses as observed in case of SARS-CoV 2 allowed us to think that the designed diagnostic assay should be able to be used at or near the point-of-care (POC) to detect a wide range of strains. Luna et al. [46] developed a non-fluorescent method for detecting the entire coronavirus genus with a detection limit of 15.7 copies/reaction and 100 copies/sample in patients with severe acute respiratory syndrome. Hardick et al. [47] evaluated a novel, portable, and near-POC diagnostic platform based on the microarray chip, the Mobile Analysis Platform (MAP), which showed a good performance in identifying the virus within an acceptable detection limit.

Current scenario in the detection of viruses
Electrochemical investigations favour significant advantages such as simplicity in design, higher sensitivity and selectivity, low cost equipment, lower power need and easy to integrate within the microfluidic devices compared to other proposed methods [48], [49], [50]. Electrochemical investigations have also demonstrated excellent applications in health care applications [51], [52]. Recently, the World has witnessed the outbreaks of diseases associated with viruses such as Ebola, MERS-CoV, SARS-CoV-1 and SARS-CoV-2 highlighting the need for rapid testing kit that can be used in the community to avoid further pandemic. A novel device with advanced instrumentation for cases such as COVID-19 is an upcoming challenge for the point-of-care diagnostic industries. However, it was observed that OECD countries have achieved a massive scaling in testing of the coronavirus that are predominately based on PCR-centralised laboratory testing than using the point-of-care devices. Moreover, reducing the sample-to-answer time is also crucial in contact tracing. Thus, reliable and high throughput testing devices continue to play the central role in containing the pandemic. Hence, in this section, we will discuss the advances made in electrochemical techniques for the detection of pathogen that can be reliable and powerful to fight against even for any future pandemics.

Immuno-assay or DNA-based assays have been the most commonly used techniques to quantify and identify the pathogens. However, the selection of assay to be used depends on various factors such as the availability of antibodies, stage of an infection and DNA sequence data, which include viral DNA, species- and strain-selective genes and toxin-producing genes. Detection of antibodies in the infected organism, either during or after infection, may offer detailed information about the infecting pathogen. The characteristics in such assays are that the antibodies are the bio-recognition element as well as the targeted moiety. Immunoassays can therefore be used to detect directly the pathogen using the available antibodies. The advantageous property of immunoassays to be used in the direct and indirect detection of pathogens via the generated antibodies and pathogen epitopes, make these techniques a flexible approach to detect the pathogens. On other hand, if the availability of antigens is limited or antigen production in the organism is significantly lower even in the presence of pathogen causing infection, then DNA-based assays can be commonly employed though these techniques require the sample to contain pathogen at the time of analysis.

The well-known bio-analytical techniques usually detect one or more components in the sample using a molecular probe as a bio-recognition element combined with the analytical system such as PCR analyzer or a plate reader. However, the robustness and sensitivity of these techniques are advantageous, and these techniques have offered time-to-results due to extensive reagent utilization in sample and complex sample preparation steps. In addition, as discussed in the previous section, PCR-based bio-analytical methods may also be affected due to contextual species in the sample, resulting in a bias and uncertainty of the measurements [53]. Considering the various limitations of traditional methods and continued requirements, real-time analysis is always a better alternative.

PCR and ELISA based techniques for the detection of pathogens have complimented the biosensors for over 25 years, wherein a selective transducer is integrated with a bio-recognition element providing a platform for the identification and quantification of the infecting pathogen. As per International Union of Pure and Applied Chemistry (IUPAC), biosensor is categorised with characteristics such as direct contact between bio-reorganisation element and transducer element to provide semi-qualitative or qualitative analytical information excluding the reagents and additional processing steps. Hence, it is a self-contained integrated device [54]. Significant and advantageous developments in biosensors have led to the fabrication of devices enabling to selective real-time detection of pathogens in various environments and matrices such as surfaces, body fluids foods, and wastewaters.

The fabrication of biosensor that can detect biological analyte purely based on their intrinsic properties, would be a difficult task. Hence, in addition to protocols that are free from sample preparation, labelled and label free protocols for the fabrication of biosensors have been proposed [55]. Labels such as enzymes and fluorescent or radioactive molecules are attached to the targeted analyte [56] and the sensor signal corresponds to the amount of labels, representing the number of bound target molecules. However, labelled protocols have some adverse drawbacks such as they are cost-intensive, sophisticated preparation facilities, trained personnel, time-consuming and they can block the active binding sites, resulting in an alteration of binding properties, thereby affecting the affinity-based interaction between the recognition elements and the target molecules [57]. Table 1 gives a summary of the utilization of some of the methods to detect different viruses, but in any case, as per literature the PCR-based analysis of viruses is the first choice.Table 2.

Table 1 – Recently used methods for viral detection

ParasiteMethodLimit of DetectionRef.
a)Measles virus (MeV)b)Rubella virus (RV)c)Human enterovirus (EV)d)Varicella-zoster virus (VZV)e)Dengue virus (DENV)f)Human parvovirus B19 (B19)g)Epstein-Barr virus (EBV)h)Human herpes virus 6Multiplex real-time RT-PCRa) MN (copies/reaction) =104MX(copies/reaction) = 94b) MN (copies/reaction) = 301MX (copies/reaction) = 81c) MN (copies/reaction) =190MX (copies/reaction) = 137d) MN (copies/reaction) = 68MX (copies/reaction) = 70e) MN (copies/reaction) = 203MX (copies/reaction) = 177f) MN (copies/reaction) = 58MX (copies/reaction) = 60g) MN (copies/reaction) = 49MX (copies/reaction) = 47h) MN (copies/reaction) = 43MX (copies/reaction) = 72MN: Monoplex; MX: Multiplex[61]
Gastroenteritis virus VN-96Electro-optical sensor∼104 viral particles ml−1[62]
African swine fever virus (ASFV)Polymerase chain reaction with a lateral flow strip1.5 × 101 copies/reaction[63]
Japanese encephalitis virus (JEV)Fluorescence molecularly imprinted sensor based on a metal-organic framework13 pmol L−1[64]
a)Tentative names: Ginger chlorotic fleck associated virus 1 (GCFaV-1b)GCFaV-1c)GCFaV-1d)GCFaV-1e)Tentative names: Ginger chlorotic fleck-associated virus 2 (GCFaV-2).f)GCFaV-2g)GCFaV-2h)GCFaV-2a)RT-LAMPb)RT-RPAc)RT-PCRd)Real-time RT-PCRe)RT-LAMPf)RT-RPAg)RTPCRh)Real-time RT-PCRa)10-5b)10-4c)10-7d)10-2e)10-3f)10-3g)100h)10-5[65]
a)Duck hepatitis A virus 1 (DHAV-1)b)DHAV-3c)Duck astrovirus 1 (DAstV-1)d)DAstV-2e)Duck reovirus 1 (DRV-1)f)DRV-2g)Tembusu virus (TMUV)h)Avian influenza virus (AIV)i)Goose parvovirus (GPV)j)Duck enteritis virus (DEV)Matrix assisted laser desorption / ionization time of flight mass spectrometrya)4.0 copiesb)7.3 copiesc)1.3 copiesd)2.1 copiese)1.3 copiesf)3.3 copiesg)3.9 copiesh)1.7 copiesi)3.4 copiesj)7.8 copies[66]
Dengue virusReverse transcription recombinase-aided amplification with lateral-flow dipstick assay10 copies/mL[67]
Porcine epidemic diarrhoea virusSYBRTM Green one-step RT-qPCR50 genome copies/5µl of extract from fecal matrices spiked100 genome copies/5µl of extract from jejunum matrices spiked[68]
Herpes simplex and varicella-Zoster virusReal-time polymerase chain reaction (RT-PCR) (Argene, BioMerieux, France) performed on an LC480 platform and isothermal amplification using a Solana HSV1 + 2/VZV assay107 copies/mL for HSV-1, HSV-2, and VZV[69]
African Swine Fever VirusCRISPR-Cas12a and fluorescence based point-of-care system1 pM[70]
Laryngotracheitis virusColloidal gold test strip based on membrane chromatography60 ELD 50/ mL[71]
Dengue and Zika virusesMultiplex RT-qPCR assaysduplex assay was 0.028 and 0.065 FFU (focus forming unit)/ml for DENV and ZIKV respectively[72]
Nervous necrosis virusA lateral flow immuno-chromatic strip105.05 TCID50/100 μL[73]
Hepatitis B virusElectrochemical DNA sensor based on nanoflowers of Cu3(PO4)2-BSA-GO1100 copies/mL for HBV-DNA[74]
Japanese Encephalitis VirusDuplex TaqMan RT-qPCR10 genomic copy[75]
a)Porcine epidemic diarrhea virusb)Porcine bocavirus (PBoV) 3/4/5Duplex real-time PCR assay based on SYBR Green Ia)10 copies/μLb)10 copies/μL[76]
Zika virusa)Colorimetric format.b)Colorimetric format.c)Electrochemical formatd)Electrochemical formata)Genosensors R1: 32 pmol L-1b)Genosensors R2: 9 pmol L-1c)Genosensors R1: 0.7 pmol L-1d)Genosensors R2: 3 pmol L-1[77]
a)Zika virusb)Chikungunya viruses -ac)Chikungunya viruses -bMultiplex RT-qPCRa)100 copiesb)5 copiesc)50 copies[78]
Porcine epidemic diarrhea virusImmuno-chromatographic strip1:50[79]
a)H1N1 of influenza A virusb)H3N2 of influenza A virusCdSe/CdS/ZnS quantum dot-linked rapid fluorescent immunochromatographic testa)28.37b)34.48[80]
Epstein-Barr virusElectrochemical detection0.46 fM[81]
a)Pseudorabies virusb)Porcine circovirus 3SYBR green I-based duplex real-time PCRa)37.8 copies/μL,b)30.6 copies/μL[82]
a)Classic swine fever virusb)Porcine circovirus 3SYBR green I-based duplex real-time fluorescence quantitative PCRa)23 copies/μLb)36 copies/μL[83]
Ebola virusRolling circle amplification of Ebola virus and fluorescence detection based on graphene oxide1.4 pM.[84]
Ebola virusMicrofluidic sample preparation multiplex0.021 pfu/mL[85]
Ebola virusElectrochemical DNA biosensor4.7nm[86]
Ebola virusRT-PCR10 molecules/μl[87]
Ebola virusFluorescently-labeled phosphorodiamidate probe pairs25pM[88]
Ebola virusReal-time reverse transcription-polymerase chain reaction10 copies per reaction[89]
Ebola virusReal-time reverse-transcription PCR assay5 × 102 viral particles per ml[90]
Ebola virusReverse-transcription-PCR (RT- qPCR)0.374 cps/μl[91]
HIVUltra-sensitive electrolyte-gated field-effect transistorHIV-1 p24 proteins at a concentration of 1 fM,[92]
HIVMolecularly imprinted electrochemiluminescence0.3 fM[93]
HIVDNA-stabilized silver nanoclusters (AgNCs)-based label-free fluorescent platform11 pM[94]
HIVElectrochemical DNA sensor, polyaniline/graphene nanocomposite1.0 X 10-16 M[95]
HIVWearable microfluidic device combined with recombinase polymerase amplification100 copies/mL[96]
HIVImpedimetric2.5 10−12 molL−1[97]
HIVLuciferase immunosorbent assay10 pg/mL and 100 ng/mL was reached for LISA and ELISA[98]
HantavirusElectrochemical immunoassayLOD of 0.14 ng mL−1[99]
DengueImmunofluorescence15 ng mL−1[100]
Colorimetric[101]
Avian influenza (AIV H5N1)Surface plasmon resonance1 pM[102]
a)classical swine fever virusb)porcine epidemic diarrhea virusc)porcine reproductive and respiratory syndrome virusd)transmissible gastroenteritis coronavirus (TGEV)Multiplex RT-PCR1 x 103 copies[103]
Influenza A (H1N1)RT-LAMR mediated colorimetric determination3 x 10-4[104]
Influenza a (pH1N1)Polydiacetylene (PDA)-based colorimetric biosensor105 TCID50 without reading device104 TCID50 with reading deviceThrough colour change from5×103∼104 TCID50 viruses[105]
Zika VirusqRT-PCR2.5 PFU/mL[106]

Table 2 – Electrochemical methods used for the detection of pathogens

Electrochemical methodsType of electrodesTargeted virusLimit of detectionRef
ConductometryAg nanofiber array electrodeBovine viral diarrhoea virus (BVDV)103 CCID/mL[132]
Cyclic voltammetryGraphene microelectrodeRotavirus10 3PFU/mL[133]
EISAu electrodeHuman influenza A virus H3N28 ng/mL[134]
ConductometryPDDA/CNT composite on Au microelectrodeSwine influenza virus (SIV) H1N1180 TCID50/mL[135]
DPVNanostructured alumina on Pt wire electrodeDengue type 2 virus (DENV-2)1 PFU/mL[136]
EIS Ferrocene methanolNanostructured alumina on Pt wire electrodeDENV-21 PFU/mL[137]
ConductometrySilicon nanowire electrode arrayHuman influenza A viruses H1N1 and H3N22.9 x 104 viruses/mL[138]
EIS; Fe(CN)63-/4-Au microelectrodeHuman influenza A virus H1N1[139]
EIS; Fe(CN)63-/4-Pt-coated nanostructured alumina membrane electrodeDENV-2, dengue virus 3 (DENV-3)0.23 PFU/mL[140]
AmperometryPolypyrrole nanoribbons on Au microelectrode arrayCucumber mosaic virus (CMV)10 ng/mL[141]
SSWV, fluorescenceAuNPs on carbon electrodeMurine norovirus (MNV)180 viruses[142]
AmperometryReduced graphene oxideRotavirus100 PFU[143]
CV, EIS; Fe(CN)6AuNPs on Au electrodeDengue virus 1–4[144]
EIS; Fe(CN)3-/4-Au interdigitated microelectrode arrayAvian influenza virus (AIV) H5N14 HAU/mL[145]
SWVAu microelectrodeNorovirus10 PFU/mL[146]
EISAu interdigitated microelectrode arrayAvian influenza virus (AIV) H5N14.2 HAU/mL[147]
EISAu electrodeHuman influenza A virus H3N21.3 x 104 viruses/mL[148]
DPVGraphene/AuNP composite on carbon electrodeNorovirus100 pM[149]
CV, EISAu electrodeNorovirus7.8 copies/mL[150]
CV, EISCarbon NPs on carbon electrodeJapanese ncephalitis virus (JEV)2 ng/mL[151]
EIS, potentiometryPEDOT film electrodeHuman influenza A virus H1N10.013 HAU[152]
Chrono-amperometryReduced graphene oxide on AuHuman influenza A virus H1N10.5 PFU/mL[153]
AmperometryPEDOT:PSS film electrodeHuman influenza A virus H1N10.015 HAU[154]
EIS;Au electrodeNorovirus1.7 copies/mL[155]
EISAu interdigitated microelectrode arrayAvian influenza virus (AIV) H5N10.26 HAU/mL[156]
EISAu interdigitated microelectrode arrayAvian influenza virus (AIV) H5N1103 EDI50/mL[157]
EISAu interdigitated electrode arrayAvian influenza virus (AIV) H5N10.04 HAU/mL[158]
ASVAuNPs on ITO microelectrodeAvian influenza virus (AIV) H5N110 pg/mL[159]
CVAu electrodeAvian influenza virus (AIV) H5N10.367 HAU/mL[160]
Chrono-amperometryCarbon electrodeHuman influenza A virus H9N216 HAU[161]
CV, EISAuNPs on ITO electrodeHuman enterovirus 71 (EV71)10 pg/mL[162]
SWVAuNPs on carbon electrodeMiddle East respiratory syndrome400 fg/mL[163]
DPVGCEInflenza virus A0.43 pg/mL[164]
LSVGCEH7N9 virus6.8pg/mL[165]
EISGold electrodeAvian Influenza virus8 ng/mL[134]
Chrono-amperometric, DPVCarbon SPEH5N1 and H1N1 virus protein8.3 pM (H5N 1) and 9.4 pM (H1N1)[166]

Advances in electrochemical sensors for the detection of viruses
Lowering the detection limit is the key for early detection of the infection and individuals are not infectious before they are normal. In such situations, electrochemical methods play a vital role. Further, this is easy to fabricate miniature devices to be useful at the point-to-care, offering immediate and reliable results. However, construction of electrochemically-based biosensors depends on the components such as transducer element, bio-recognition elements, and measurement formats.

In case of an electrochemical biosensor, transducer element is a cell consisting of three electrode system (potentiostat) or a two electrode system (conductometry and electrochemical impedance spectroscopy) in which much importance relies on the working electrode [58], [59], [60]. The working electrode can be fabricated with semiconducting and conducting materials ranging from metals to non-metals such as carbon, and using the materials of various sizes from bulk materials to micro and nano-structures. The electrode properties and structures affecting the performance of the electrode in terms of selectivity and limit of detection are dependent on the materials used, fabrication methods employed and the design approach.

Various metal-based electrodes consisting of gold and platinum are used as biosensors [107], [108], [109]. For instance, thick metal surface or a thin film metal electrode have been fabricated by cutting or traditional micro-fabrication using physical vapour deposition and screen printing techniques [110], [111]. In addition, ceramic electrode (consisting of polysilicon, TiO2, and indium tin oxide) and polymer electrodes (with advantageous properties of stability, biocompatibility and tuneable electric conductivity) have also been used in the fabrication of electrodes [112], [113]. However, the selection of materials for the fabrication of electrochemical sensors, especially while detecting pathogens requires expert skills. Since significant aspects of electrochemical sensor performance such as rate of heterogeneous electron transfer, double layer capacitance, nature of coupling chemistry required immobilising the bio-receptors may be affected. Furthermore, since Faradic current is dependent on the active electrode surface area, increasing the surface area improves the sensitivity as well as controls the background current. A simple and effective mode to increase surface area is to use the nanomaterial and the composites. This would also facilitate easy immobilization of bio-receptors, thus increasing the sensitivity in a wider dynamic range, thereby allowing higher collision frequency between the antigens and the antibodies [114].

Elevating the target and/or selective binding based on enzymes or antibodies are the principal needs for biomolecular recognition. Limited stability of these complex materials are often accompanied with multifaceted protocols and specific handling protocols. For virus detection receptors to be reused they can mimic antibodies recognition properties that are favourable, especially in health care systems. Hence, recent efforts on molecular imprinting strategies have evolved significantly allowing the fabricated sensor to mimic immunological interactions [115].

In molecular imprinting, the first step involves the interaction between cross-linking agents and the monomers in a suitable solvent with the templates; then following the arrangement of formed molecular assemblies by PCR around the template molecules, and finally removing the templates leaving behind the analyte selective binding moieties. Recently, extensive studies based on molecularly imprinted polymers have detected a wide range of species targeting proteins, cells and viruses [116], [117]. These included different polymerization strategies such as surface imprinting (2D) and bulk imprinting (3D). To perform the bulk imprinting, the respective template was directly added to a monomer mixture and the hydrogels formed with 3D matrices could offer less restricted diffusion pathways [118], [119].

Diverse examples of hydrogels used to imprint viruses are available in the literature [120], [121], [122]. On other hand, surface imprinting can be achieved by attaching the template to the supporting material or by a thin polymer film decoration. These methods can be carried out using soft lithography, self-assembly or by core-shell particles via immobilized templates. On the other hand, the traditional imprinting techniques have focussed on materials that can favour small molecular templates. Key issues such as solubility, size, fragility, and compositional complexity are to be considered while imprinting the viruses.

The fabrication of electrodes using carbon materials has advanced significantly and classical carbon-based sensors are mainly glassy carbon, carbon fibers and pyrrolytic graphite [123], [124]. Most of the carbon-based nanomaterials have many advantageous properties such as higher electro-catalytic, adsorption bio-compatibility and fast electron transfer rate [125]. For sensor applications, carbon nanotubes and graphene have been investigated as these can be directly incorporated into a biological sensor following the simple drop casting, growing the material directly on the substrate, co-depositing with metal nanoparticles and then using them in field effect transistors [126].

Wasik et al. [127] developed an electronic biosensor based on heparin functionalized carbon nanotubes for the detection of dengue virus. Among the heparin, heparin sulfate proteoglycans have been used as receptors for dengue virus during the infection of Vero cells to detect the lowest concentration up to 8.4×102 TCID50/mL ∼8 dengue virus/chip. This method was chemiresistor functionalized with heparin instead of using an antibody for the detection of dengue virus.

The single walled carbon nanotube network can be synthesised by self-assembly on gold electrode lithiographically where the primary amine linker, 1 –pyrenemerhylamine can be adsorbed on the single walled carbon nanotubes that are cross-linked with heparin carboxyl groups and used for detecting the dengue virus. Selectivity of the biosensor was evaluated [127] using influenza virus H1N1 as the negative control.

Navakul et al. [128] demonstrated electrochemical biosensor based on impedance spectroscopy using gold electrode deposited with graphene oxide to detect in the limit of 0.12 pfu/ml. Schematic representation of the deposition of graphene oxide polymer onto the surface of gold electrode is shown Fig. 6 . The specificity of fabricated electrode was assessed since the copolymer was electrically conductive due to the presence of graphene oxide.

Further, the negative charge of oxygen atom in graphene attracts the dengue virus particles with a positive potential. Joshi et al. [129] demonstrated a method using thermally reduced graphene oxide deposited onto indium tin oxide/glass electrodes for quantitative determination of influenza virus H1N1 as a label free electrochemical immuno-sensor using impedance spectroscopy. The detection limit reported by this method was 26 and 33 pfu/mL for saline and diluted saliva, respectively.Fig 7. Fig 8. Fig 9. Fig 10.

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Fig. 6
A schematic representation of the fabrication of chemiresistor used for the detection of dengue virus (from Ref. 127).
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Fig. 7
Schematic representation of the preparation of GO-polymer on gold electrode for DENV detection (from Ref. 128)
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Fig. 8
(A) Schematic illustration to display the synthesis route of TrGO using Shellac biopolymer; (B) Schematics of the proposed thermally-decomposed reduced graphene oxide (from Ref. 129)
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Fig. 9
Nano based material for sensor application
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Fig. 10
Schematic image of the fabricated AIV detection biosensor. (From Ref. 179)

Bhattacharya et al. [130] demonstrated layer-by layer assembled functionalized carbon nanotubes for interaction with antiviral antibodies and avian metapneumo virus. A form of resistor was developed patterning onto gold electrode on a Si/SiO2 substrate onto which layer-by-layer assembly of carbon nanotubes were built. The poly(diallyldimethylammonium chloride), poly(styrene sulfonate), and functionalized single-walled carbon nanotubes were absorbed onto the surface electrostatically to form the multilayer films. The interaction and immobilization of viral antibodies between the electrode was enhanced using poly(L-lysine), further capturing the antibody specific antibodies. This device was able to detect the change in conductivity with an antigen of 102 TCID50/mL.

Fu et al. [131] developed chemiresistive biosensor based on carbon nanotubes for detecting avian influenza virus H5N1. In these sensors, long nanotubes (>5 _m) were placed between the inter-digitised metal electrodes such that individual nanotubes connect the electrodes and nanotubes were functionalized with DNA probe sequences non-covalently attached to the sidewalls. These functionalized-nanotube sensors reliably detected the complementary DNA target sequences of AIV H5N1 within a concentration range between 2 pM and 2 nM in 15 min at room temperature.

Application of Nanotechnology
In recent years, outstanding achievements in nanotechnology have allowed novel materials to steadily intensify their new horizons across the globe [167], [168], [169], [170], [171], [172], [173], [174], [175].

Flexibility and expendability of the nanomaterials are prowling in biomedical areas as some have almost reached commercialization, especially for viral detection [176]. In addition, such advances have led nanotechnology with remarkable opportunities for the detection and diagnosis of viral infections since these materials have unique properties compared to their bulk materials and have significantly enlarged surface-to-volume ratio. Surface properties, size and composition of nanomaterials can be engineered to develop robust materials with superior luminescent and electrochemical properties.

Metals and metal oxide nanoparticles in the size range of 1–100 nm are significantly suitable to fabricate biosensors due to their noticeable increase in surface area to volume ratio since the size of the material decreases. For instance, in biomedical area, unique optical and electric properties of gold nanoparticles have been well established for the detection of viruses. Shariati et al. [177] proposed label free detection of human papilloma virus based on gold nanotubes using the electrochemical impedimetric technique.

In this study, external electric field applied allowed the preferred orientation of the negatively charged DNA oligonucleotide to increase the sensing response via controlled hybridization and immobilization of the sequence onto gold nanotubes surface. This biosensor has shown a lower detection limit of 1 fM in the linear range of 0.01 pM to 1 µM.

Lee et al. [178] developed an electrochemically based label-free avian influenza virus detection method using multi-functional DNA structure on a porous (p) AuNPs-modified electrode. The proposed DNA 3 way-junction/pAuNPs based detection method can be applied for multiple-target detections as a valuable biosensor for determining the pathogen subtype in one platform or one target detection using the dual detection method with high reliability.

Zhao et al. [179] demonstrated a stable electrochemical method for the detection of hepatitis B virus based on nanoflowers of Cu3(PO4)2-, gold nanoparticles by amplifying the signals using two aptamers. The fabricated sensor showed excellent binding points along with adaptive outline for the amplification of signal and biocompatibility. The use of 3D nanoflowers was advantageous to increase the surface area, reaction kinetics, carrier immobility, and charge transfer.

The structural features such as petals and their dimensions were controlled by synthesizing a new organic and inorganic framework based on CuSO4·5H2O, bovine serum albumin and graphite oxide. The extra binding sites were further obtained using gold nanoparticles to enhance the interaction between electrode and thiol-functionalized Aptamer-1. The detection limit was 1100 copies/ml for hepatitis B virus DNA in a dynamic linear range of 1.10×103 to 1.21×105 copies/mL.

Silver nanoparticles offer advantages of simple preparation and have good binding ability with biomolecules that can offer excellent opportunities for the fabrication of electrochemical biosensors. Khristunova et al. [74] developed an electrochemical immuno-sensing technique for the detection of antibodies to tick-borne encephalitis (TBEV).

Thiolation and glutarization of the composite electrode containing gold-carbon was performed prior to covalent immobilization of the antigen onto the surface of the electrode. Further, measurement of released silver species from the silver nanoparticles containing bio-conjugates with antibodies to TBEV was the basis of assay of this method. The proposed method was suitable to quantify antibodies to TBEV in the range of 100-1600 IU mL-1, with a LOD of 90 IU mL-1.

Nanomaterials based on graphene are advantageous due to their outstanding chemical, mechanical, thermal and electronic properties and these are quite predominant to design the biosensors for DNA detection due to their enhanced affinity towards single-stranded DNA by the hydrophobic interaction and π-π stacking [180]. Graphene can be used as a substrate to interface with different cells and biomolecules, which is beneficial to improve its biocompatibility, solubility and selectivity

Li et al. [181] proposed DNA-assisted magnetic reduced graphene oxide-copper nanocomposite for the sensitive detection of hepatitis C virus DNA in the linear range of 0.05 to 10 nm with a detection limit 405 pM. The copper ions assisted for accelerated oxidation of o-phenylenediamine producing 2, 3-diaminobenazine from which electrochemical signals were obtained for the characterisation of hepatitis C virus DNA. Wang et al. [182] developed a sandwich type of electrochemical immunoassay for the sensitive detection of avian leukosis virus subgroup J using graphene quantum dots and apoferritin- encapsulated Cu nanoparticles. Differential pulse voltammetry was used to detect the released Cu from apoferritin cavity from the assembly.

The electric signals increased effectively owing to the huge surface area of graphene quantum dots accommodating a considerable amount of antibodies loading. Here, Cu-apoferritin nanoparticles were responsible to increase the loading of electroactive probe to further amplify the signals. The method could detect avian leucosis virus in the range of 102.08 to 104.50 TCID50/mL with a detection limit of 115 TCID50/mL.

Acute and chronic diseases caused by various viruses pose real threat to the human. These include smallpox, influenza, diarrhea, AIDs, hepatitis and polio, which have devastating effects that need initial cautionary system for the recognition and detection of the virus. Use of nanotechnology in the initial recognition of viruses is interesting and eye catching. Literature reports suggest the application of nano-biosensors, immobilization schemes and immuno-sensing for the detection of various viruses.

The advantageous applications of nanomaterials in addition to electrochemical nano-biosensors have been useful for the development of several diagnostic procedures in medical arena. Their applications are wide-spread in the quantification of numerous clinical biomarkers, evaluation as well as follow up after the illness. At present, graphene, magnetic nanoparticles, carbon, gold silica, and quantum dots have provided a subtle and precise strategy in bio-sensing.

The highly promising ability of a biosensor using electrochemical nanosensor has to address many challenges, one of which is to establish itself at the point-of-care. In addition, the profit guarantee in bio-sensing relies largely on the selection of an appropriate nanomaterial to function as a useful biosensor. Further, the risk of errors and mistakes in the detection of virus are critical when considering the immobilization method of concerned nanomaterial.

Reusable and portability are the other two factors with properties such as easy discrimination of viruses along with sensitivity and selectivity level, which indeed need intense efforts in addition to lifetime of the assay. In summary, nanotechnology with much greater advances in the near future will certainly be a breakthrough in biomedical area to check and stop the viral diseases to provide healthy life.

In recent years, personalized medicine and digital health monitoring is becoming increasingly attractive and this tremendous potential has now become realistic due to the fabulous advances in skin interfaced wearable sensors. These sensors interface with the skin in a wide range of sizes from cellular level down to molecular level and these hold the capability for therapeutic and diagnostic functions with excellent precision, continuity and expediency. In addition, the new opening of adding artificial intelligence and integrated cloud-based technologies would enhance the utilization of smarter healthcare systems.

These devices compared to the traditional healthcare systems, can collect non-invasive data from the human body to provide an insight into both fitness monitoring and medical diagnostics along with keeping a track of molecular biomarkers of the human system. For such sensor devices, electrochemical detection would probably be one of the most fitting techniques due to its easiness in miniaturization, and low electric power consumption. This review will not dare to dwell into these details as it is beyond the scope of the subject though some recent publications address these issues [183], [184], [185], [186], [187], [188], [189], [190], [191].

Rising test strategies for COVID-19 diagnosis
WHO in such current pandemic situation has given a suggestion for the development of techniques, which are rapid in response, especially that are based on nucleic acid and protein test formats [192]. These developed techniques should offer advantages for use in short-term at the point-of–care. Effectiveness for tracking and surveillance can be enhanced using serological test for the detection of protein. Further, emphasis on cost effectiveness, lowering the burden on clinical and central laboratories by easing the operation must be performed [193]

SHERLOCK method for the detection is one of the nucleic acid-based methods that has emerged and has all the potentials to be applied for the detection of SARS-CoV-2. Based on CSISPR method, a gene-editing tool used for RNA sensing using variants of Cas9, known as Cas13a ribonuclease. The process works by targeting the virus RNA followed by reverse transcription to DNA and isothermal amplification.

Further DNA is transformed back to RNA where it interacts with Cas 13a. The targeted molecule activates Cas13a allowing the cleavage with fluorescent probe that yields the signal. Hou et al. [194] developed an isothermal, CRISPR-based method for the detection of SARS-CoV-2.

Detection of viral spike proteins and antibodies generated in the patients after the infection is another approach for the diagnosis of SARS-CoV-2. In detecting the coronavirus, antibodies studies have shown that S proteins from SARS-CoV-2 have greater reactivity. On other hand, an enzyme linked immuno-sorbent assay (ELISA) for detecting immuno-globins G and M in serum of the infected persons was demonstrated [195].

The studies used for nucleocapsid protein Rp3 from SARS-CoV-2 that has 90% similarity with SARS viruses. The results recorded on day 0 showed 50% positive for IgM and 80% for IgG, further increasing to 80% and 100% on day 5 [196]. The method has flexibility of sample such as blood, fecal and respiratory organs.

Considering the on-site testing that plays an important role in point-of-care that provides many advantage in which the key point is the on-spot detection avoiding the transport of samples to laboratories. One such a method proposed is the lateral flow assay, which is still under development for SARS-CoV-2. This method utilizes a paper strip coated with gold nanoparticles functionalized with antibodies. Simple colour change due to clustering through plasmon banding of gold nanoparticles are observed to derive the results. Such methods have been used for MERS-CoV, but challenges such as single usage and low sensitivity are yet to be addressed and further research is needed to be done on the amplification of readouts.

Another approach used is the designing of Microfluidic devices using a small chip consisting of micro-channels for the reaction. Such microfluidic device-based smart phone for detecting antibodies of sexually transmitted diseases has been demonstrated [197]. Though the sensitivity of this method is in the range of 90–100%, attempts to use this technology for SARS-CoV-2 are not still available and if achieved, it would be a viable option for detecting specific proteins and nucleic acid for SARS-CoV-2.

RT-PCR devices have faced major challenges such as requirement of skilled and trained staff, infrastructure, and it take at least 6-24 h for the results. Hence, these techniques are not efficiently used in conditions of rapid screening in crowded places where huge numbers of samples have to be tested. However, portable RT-PCR devices can be another option for SARS-CoV-2, since portable RT-PCR has been used in plant pathogenesis.

These devices are portable and upon the addition of a target viral RNA, it can detect the host biomarkers. As the early investigations of SARS-CoV-2 have shown S and N proteins are predominant, nucleic acids of these can be targeted in these tests. The advantage of this device is that the analysis of RNA takes place in less than 2 h on site avoiding the need for sample transportation to laboratories [198].

Chemiluminesence immuno-assay is one such an approach that has been popularly used in the detection of infectious diseases. Recent studies have demonstrated the application of this method for SARS-CoV-2 [199], [200], [201]. Cai et al. [200] demonstrated a peptide-based magnetic chemiluminesence enzyme immuno-assay for detecting SARS-CoV-2 antibodies against ORF1a/b, N and S proteins.

The positive rate for this method was 71.4% for IgG and 57.2% for IgM. In any case, the approach based on RT-PCR has the first line of defence in the diagnostic test of SARS-CoV-2. Efforts are being made to develop new serological tests since there is an urgent need for efficient on-site and multiplex platforms. Technologies are still in the developmental stage and more focus should be deployed on creating clear communication network.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605744/


More information: Ashwin Ramachandran et al, Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.2010254117

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