Lateral Flow Strip Biosensor Research Articles

An integrated self-heating recombinase polymerase amplification lateral flow strip biosensor for quantification of enterohemorrhagic Escherichia coli O157:H7

The early and precise detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7, a major foodborne pathogen, is crucial to prevent outbreaks and ensure food safety. However, traditional diagnostic methods are often time-consuming, labor-intensive, and require sophisticated laboratory facilities, making them less feasible for point-of-care testing. Here, we present a novel, self-heating recombinase polymerase amplification (RPA)-based integrated lateral flow strip biosensor for the efficient detection of EHEC O157:H7. The self-heating mechanism based on calcium oxide provides a stable and optimal external temperature for RPA, avoiding the use of external power sources or complex temperature control devices. In addition, the color intensity of the test line of the lateral flow strip can be uploaded to the ImageJ software by taking photos, and the quantitative detection of EHEC O157:H7 can be realized by analyzing the gray value. Our results demonstrate that this biosensor has a visual detection limit of 1 × 102 CFU/mL and an analytical limit of detection of 36.23 CFU/mL for EHEC O157:H7, offering rapid detection within 30 min. Furthermore, the biosensor displayed exceptional specificity and exhibited high accuracy. The RSD of three varying concentrations were respectively 5.39 %, 3.85 % and 4.82 %.

Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system

Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.

Absolute Quantification of Serum Exosomes in Patients with an SERS-Lateral Flow Strip Biosensor for Noninvasive Clinical Cancer Diagnosis.

Exosomes (exos) widely existing in body fluids show great potential for noninvasive cancer diagnosis. Quantitative analysis of exos is traditionally performed by targeting specific exosomal surface proteins, but it is often imprecise due to the common expression of exosomal proteins and subtle expression differences between different cancer subtypes. Herein, we report quantitative surface-enhanced Raman spectroscopy (SERS) of serum exos through a combination of a paper-based lateral flow strip (LFS) biosensor with multivariate spectral unmixing analysis rather than simply quantifying exosomal proteins. Our SERS-LFS biosensor enables absolute quantification of two different serum exos with a limit of detection down to ∼106 particles/mL for both exos. We further exemplify the application of this strategy in quantitative dual-plex detection of serum exos from breast cancer patients. We find that human epidermal growth factor receptor 2+ (HER2+) and luminal A breast cancer patients undergoing no surgery are enriched in serum exos derived from SKBR-3 cells and MCF-7 cells (denoted as SKBR and MCF exos), respectively. The surgical treatment of these breast cancer patients accompanies an obvious decrease of either SKBR or MCF exos in the serum. These results suggest the great potential of the combination of the SERS-LFS biosensor and multivariate spectral unmixing for breast cancer subtyping and therapeutic surveillance with the powerful quantitative capability of exos in clinical samples.

Lateral Flow Strip Biosensors for Foodborne Pathogenic Bacteria via Direct and Indirect Sensing Strategies: A Review.

Rapid and sensitive detection of foodborne pathogenic bacteria is particularly important for the prevention and control of foodborne diseases. The lateral flow strip biosensor (LFSB) is one of the most promising point-of-care detection tools and has been widely used in food safety monitoring. This review introduces recent advances in the detection of foodborne pathogenic bacteria using LFSBs. According to different bacterial biomarkers, we summarize the direct and indirect sensing strategies of bacterial LFSBs. The direct sensing strategies for whole bacterial cells are divided into antibodies, antibody alternatives, and label-free according to the recognition elements. The indirect sensing strategies refer to the detection of bacterial nucleic acids and metabolites. Next, we compare and discuss the applications of direct and indirect sensing strategies. Finally, the existing challenges, future perspectives, and development directions are discussed, which will facilitate the theoretical innovation and practical application for bacterial LFSBs.

A lateral flow strip biosensor platform based on cascade nucleic acid amplification technology for ultrasensitive detection of OSCC-associated salivary MicroRNA

Oral squamous cell carcinoma (OSCC) is the well-known malignancy and poses a serious threat to human health with high morbidity and mortality. Early detection and treatment can improve the recovery rate and reduce complications of OSCC. Therefore, we designed a lateral flow strip biosensor platform (HRCA-strip) based on the cascade nucleic acid amplification technology (HRCA) for colorimetric analysis of OSCC-associated has-microRNA 31-5p (miRNA 31). In this work, the target miRNA 31 mediated the formation of the sandwich complex structure on the surface of magnetic beads (MBs). Then, the sandwich complex structure could activate cascade amplification reaction between hybridization chain reaction (HCR) and rolling-circle amplification (RCA) to generate numerous G-quadruplex structures. The G-quadruplex structures combined with hemin to form hemin/G‐quadruplex horseradish peroxidase‐mimicking DNAzyme (H/G-HRP mimic enzyme) which were enriched on the T-line and catalyzed the oxidation of chromogenic substrates to generate colorimetric signal on the strip. The HRCA-strip platform could achieve highly sensitive and specific miRNA 31 detection with the limit of detection (LOD) as low as 3.21 fM. Moreover, the designed HRCA-strip platform also enabled portable detection of miRNA 31 in clinical sample which might show good potential for early clinical diagnosis of OSCC.

Lateral flow strip biosensor based on streptavidin-coated gold nanoparticles with recombinase polymerase amplification for the quantitative point-of-care testing of Salmonella

Salmonella is a widespread and highly infectious biological contaminant that causes zoonotic diseases. Most detection methods are qualitative, while quantitative methods are time-consuming and rely on large analytical instruments. Therefore, the development of a rapid, simple, and quantitative assay is important for the prevention and control of infection at the early stages of an outbreak. To address this requirement, we developed a lateral flow (LF) strip biosensor based on streptavidin-coated gold nanoparticles (StreptAv–AuNPs) combined with recombinase polymerase amplification (RPA) for the quantitative point-of-care testing of Salmonella enterica serotype Enteritidis (S. Enteritidis). StreptAv-AuNPs were simple to synthesise and bound firmly to DNA with end-modified biotin. RPA could greatly reduce detection time. A smartphone was used to collect images of the LF strip and input them into a laptop for quantitative analysis. The entire process was completed in less than 40 min and did not require large laboratory equipment or analytical instruments. A limit of detection of 5.5 × 10−13 M and 91.4 CFU/mL was achieved for synthetic S. Enteritidis DNA and cultured S. Enteritidis samples, respectively. In the reproducibility assay, RSD values of 5.88%, 5.13%, and 4.45% were achieved for the three different concentrations of samples. This method has the advantages of low cost, high precision and rapid quantification, it is also suitable for the detection of various DNA-containing analytes.

Development of a Nanoparticle-based Lateral Flow Strip Biosensor for Visual Detection of Whole Nervous Necrosis Virus Particles

Effective analysis of pathogens causing human and veterinary diseases demands rapid, specific and sensitive detection methods which can be applied in research laboratory setups and in field for routine diagnosis. Paper lateral flow biosensors (LFBs) have been established as attractive tools for such analytical applications. In the present study a prototype LFB was designed for whole particles (virions) detection of nodavirus or fish nervous necrosis virus. Nodavirus is an important threat in the aquaculture industry, causing severe economic losses and environmental problems. The LFB was based on polyclonal antibodies conjugated on gold nanoparticles for signal visualization. Brain and retinas from fish samples were homogenized, centrifuged and the supernatant was directly applied on the LFB. Formation of a red test line was indicative of nodavirus virions presence. Nodavirus visual detection was completed in short time (30 min). Key factors of the LFB development influencing the assays’ detection limit were characterized and the optimum parameters were determined, enabling increased efficiency, excluding non-specific interactions. Therefore, the proposed LFB assay consists a robust, simple, low cost and accurate method for detection of nodavirus virions in fish samples. The proposed biosensor is ideal for development of a commercial kit to be used on aquaculture facilities by fish farmers. It is anticipated that disease monitoring and environmental safety will benefit from the simplification of time consuming and costly procedures.

Visual detection of kanamycin with DNA-functionalized gold nanoparticles probe in aptamer-based strip biosensor

Kanamycin has been widely used to treat human and animal diseases. The excessive use of kanamycin causes its accumulation in animal-derived foods, and eventually threats human health. In the present study, we develop a lateral flow strip biosensor for fast and sensitive detection of kanamycin. The strip biosensor combines the easy separation of magnetic microspheres (MMS) with target-mediated chain displacement of single-stranded DNA and the capture of the visible DNA-functionalized gold nanoparticles (AuNPs) probe. The presence of kanamycin can competitively bind to the aptamer and release cDNA to the supernatant. The concentration of free cDNA, which is the direct target of the strip, is proportional to the concentration of kanamycin. The capture of DNA-functionalized AuNPs on the test zone of the strip through cDNA-induced hybridization provides a visual detection signal. The assay can be completed within 20 min. The visual detection limit by naked eyes of the strip is 50 nM. A linear detection range of 5–500 nM is derived for quantitative determination, with the detection limit of 4.96 nM (S/N = 3). This lateral flow strip biosensor can quickly and sensitively detect kanamycin in different food samples, which holds great application potential in medicine and daily life.

Visual simultaneous detection of single nucleotide polymorphism of tumor susceptibility gene and marker alpha-fetoprotein based on double-labeled colloidal gold probe with lateral flow strip biosensor

Liver cancer is one of the most common cancers in the world. The gene CYP1A1 is closely related to the occurrence of cancer, and the tumor marker alpha-fetoprotein (AFP) is also one of the important indicators for liver cancer screening. Therefore, detection of these two substances is of great significance. In this paper, a test strip biosensor capable of simultaneously detecting cancer susceptibility gene CYP1A1 and AFP has been established. There were two detection lines in the strip biosensor, one was used for qualitative detection of susceptible gene by base complementary pairing, and the other was used for semi-quantitative detection of AFP by antigen-antibody reaction. Finally, signal amplification was achieved by horseradish peroxidase catalyzing substrate 3-amino-9-ethylcarbazole. Results showed that under the optimized conditions, the strip biosensor had a good linear relationship (R2 = 0.987) when AFP concentration was in the range of 20–250 ng/mL, and the other hundred times of proteins had no obvious interference to the detection, indicating that the method presented good selectivity. The detection of susceptibility gene CYP1A1 and AFP in real blood samples by the strip was in accordance the standard method, which showed that the method had good accuracy and could be used for qualitative judgment of SNP locus base type of susceptibility gene and semi-quantitative detection of tumor marker in real sample, so it had a good application potential.

Development of a lateral flow strip biosensor based on copper oxide nanoparticles for rapid and sensitive detection of HPV16 DNA

Human papillomavirus (HPV) type 16 is widely distributed in population, which is responsible for more than 50% of cervical cancers. A point-of-care testing for HPV16 is critical for early diagnosis and decisions regarding patient care. In this work, a novel lateral flow strip biosensor (LFSB) has been established by a simple, low-cost and green synthetic procedure for the detection of HPV16 DNA. Copper oxide nanoparticles (CuO NPs) were used as colored tags in the LFSB for the first time. In a typical experimental procedure, CuO NPs were sequentially functionalized with streptavidin and biotinylated capture DNA to form the CuO Capture probes. In the present of HPV16 DNA, the CuO Capture probes would capture the target, forming a characteristic brown band due to the second base pairing with the test DNA probes on the test zone of LFSB, which enabling the visual detection. A portable strips reader achieved the quantitative detection. The optimized CuO-based LFSB was capable of detecting the target over its concentration ranged from 5 nM to 100 nM with a limit of detection of 1.0 nM, and the whole detection process could be completed within 20 min. The CuO-based LFSB shows a great promise and versatile opportunities in point-of-care diagnosis of genetic diseases with good reproducibility and stability.

The sensitive detection of ODAM by using sandwich-type biosensors with a cognate pair of aptamers for the early diagnosis of periodontal disease

This research aims to develop biosensors which could diagnose periodontal diseases in early phases and predict the illness stage of patients, in order to give them adequate treatment timely. Human odontogenic ameloblast-associated protein (ODAM) is considered to be a potential biomarker for periodontal diseases, based on high correlation between the level of ODAM in gingival crevicular fluid (GCF) and the degree of periodontitis. Many aptamers, including a cognate pair of aptamers which can bind to the different sites of ODAM, were successfully screened in a very stringent condition employing saliva as a counter target through the graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX). For the characterization of the aptamer candidates, GO-based fluorescence resonance energy transfer (GO-FRET) and surface plasmon resonance (SPR) assays were conducted. The sandwich-type binding of a cognate pair of aptamers to ODAM was additionally confirmed by employing circular dichroism (CD) and magnetic beads-based fluorescence imaging methods. The resulting cognate pair of aptamers, OD64 and OD35, were found to have their dissociation constant (Kd), 47.71 nM and 51.36 nM, respectively. The minimum detectable concentrations of a sandwich-type SPR biosensor were found to be 0.24 nM and 1.63 nM, respectively, for both buffered and saliva samples. Finally, using this cognate pair of aptamers, a sandwich-type lateral flow strip biosensor was successfully realized. This research shows the potential for implementation of a cognate pair of aptamers on point-of-care biosensors which enables simple, rapid, and non-invasive saliva-based diagnosis of periodontal-related diseases that can overcome current diagnostic methods and improve health care system.

Applicability of biological dye tracer in strip biosensor for ultrasensitive detection of pathogenic bacteria

Here, a facile, label-free and sensitive lateral flow strip (LFS) biosensor for foodborne pathogens was established relying on the innovative introduction of Gram staining and the direct immunoreaction. Target bacteria can be directly marked with crystal violet (CV) by one-step staining which is superior to traditional signal marking techniques in LFS assay, and the method’s selectivity can be guaranteed by high-specificity monoclonal antibody. With Salmonella Enteritidis (S. Enteritidis) as a model target, this protocol can selectively detect 80 CFU mL−1S. Enteritidis within 11 min in the optimized conditions. Moreover, with Listeria monocytogenes as another model target, the biosensor shows a high universality for detections of both gram-negative and gram-positive bacteria. The unexpected applicability of biological dye tracer in strip biosensor reveals that the biological dye can be a potential tool serving as a universal signal tracer for pathogenic microorganisms in food safety monitoring and early clinical diagnosis.

DNA Probes for Implementation of Multiple Molecular Computations Using a Lateral Flow Strip Biosensor as the Sensing Platform.

A series of advanced logic circuits have been successfully constructed to perform multiple arithmetic functions using a lateral flow strip biosensor as the sensing platform, including half adder, half subtractor, multiplexer, demultiplexer, and keypad lock. In each circuit computing, the computing elements are the DNA probes, which can implement the DNA assembly process to generate the output single-stranded nucleic acid. The strip biosensor is used as the sensing platform to distinguish the output of the logic gates. The distinctive advantage of our proposed biocomputing system is that the logic events can be transformed into the red bands on the test zones, which can be observed by the naked eye. The logic system with convenient operation, rapid response, and cost-effectiveness features will facilitate the portable analysis at ambient temperature without resorting to any instrumentation. The versatility and power of the logic computation demonstrated here indicate its great potential in intelligent point-of-care (POC) disease diagnosis and on-site environmental monitoring.

Magnetized carbon nanotubes for visual detection of proteins directly in whole blood

The authors describe a magnetized carbon nanotube (MCNT)-based lateral flow strip biosensor for visual detection of proteins directly in whole blood avoiding complex purification and sample pre-treatments. MCNT were synthesized by coating Fe3O4 nanoparticles on the shortened multiwalled carbon nanotube (CNT) surface via co-precipitation of ferric and ferrous ions within a dispersion of shorten multiwalled CNTs. The antibody-modified MCNTs were used to capture target protein in whole blood; the formed MCNT-antibody-target protein complexes were applied to the lateral flow strip biosensor, in which a capture antibody was immobilized on the test zone of the biosensor. The captured MCNTs on the test zone and control zone were producing characteristic brown/black bands, and this enabled target protein to be visually detected. Quantification was accomplished by reading the intensities of the bands with a portable strip reader. Rabbit IgG was used as a model target to demonstrate the proof-of-concept. After systematic optimizations of assay parameters, the detection limit of the assay in whole blood was determined to be 10 ng mL−1 (S/N = 3) with a linear dynamic range of 10–200 ng mL−1. This study provides a rapid and low-cost approach for detecting proteins in blood, showing great promise for clinical application and biomedical diagnosis, particularly in limited resource settings.

Development of quantitative immunochromatographic assay for rapid and sensitive detection of carbohydrate antigen 19-9 (CA 19-9) in human plasma

A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle (GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that has been associated with cancers (such as pancreatic and colorectal cancers) and various non-cancerous diseases. The principle is based on sandwich-type immunoreactions between gold nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9 conjugates and the components of the running buffer were optimized to obtain the best sensitivity and reproducibility of the assay. The detection limit of the assay was determined to be 5UmL−1 (S/N=3) with a linear range of 5UmL−1–100UmL−1. CA 19-9 concentrations in healthy human and pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative immunochromatographic assay (QIA), and the results were in accordance with that obtained with enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical application and biomedical diagnosis, particularly in limited resource settings.

Lateral flow assay for carbohydrate antigen 19-9 in whole blood by using magnetized carbon nanotubes.

The authors describe a rapid, low-cost and sensitive approach for the determination of carbohydrate antigen 19-9 (CA 19-9) in whole blood by using magnetized carbon nanotube (MCNT) and lateral flow strip biosensor (LFSB). MCNTs were synthesized by depositing magnetite (Fe3O4) nanoparticles on multiwalled carbon nanotube (CNT) via co-precipitation of ferric and ferrous ions within a dispersion of shortened multiwalled CNTs. Antibody against CA 19-9 (Ab1) was covalently immobilized on the MCNTs and were used to capture CA 19-9 in blood. After magnetic separation, the formed MCNT-Ab1-CA 19-9 complexes are applied to the LFSB, in which a capture antibody (Ab2) and a secondary antibody (Ab3) are immobilized on the test zone and control zone of the LFSB, respectively. The captured MCNTs on the test zone and control zone are producing characteristic brown bands, and this enables CA 19-9 to be visually detected. Quantitation is accomplished by reading the intensities of the bands with a portable strip reader. Under optimized conditions, the assay has a detection limit as low as 30 U⋅mL-1 of CA19-9 in blood. This is below the cutoff value (37 U mL-1) of CA 19-9. The assay duration for blood samples is 35 min. In our perception, the assay represents a rapid and low-cost tool for rapid determination of CA19-9 in blood that holds promise for clinical applications, particularly in limited resource settings.

A signal-enhanced lateral flow strip biosensor for ultrasensitive and on-site detection of bisphenol A

ABSTRACT A signal-enhanced lateral flow strip biosensor has been constructed for ultrasensitive and on-site visual detection of bisphenol A (BPA). The signal amplification principle is based on the capacity of binding a large number of antibody-assembled gold nanoparticle (AuNP–antibody) conjugates of the poly amidoamine (PAMAM), which bears a multitude of amino groups. This signal-enhanced method could achieve BPA detection at 10 ppb based on naked eye visual observation, which represents an at least 50-fold improvement of the sensitivity of a traditional method without PAMAM. Moreover, good linearity was observed in the range from 2 to 100 ppb after quantitative analysis using the ImageJ software. This signal amplification method could be adopted as a potential generous technique in different fields.

Gold nanocage-based lateral flow immunoassay for immunoglobulin G.

The authors describe a gold nanocage-based lateral flow strip biosensor (LFSB) for low-cost and sensitive detection of IgG. This protein was used as a model analyte to demonstrate the proof-of-concept. The method combines the unique optical properties of gold nanocages (GNCs) with highly efficient chromatographic separation. A sandwich-type of immunoreactions occurs on the GNC-based LFSB which has the attractive features of avoiding multiple incubation, separation, and washing steps. The captured GNCs on the purple test zone and control zone of the biosensor are producing characteristic purple bands, and this enables IgG even to be visually detected. Quantitatation was accomplished by reading the intensities of the bands with a portable strip reader. The LFSB fabrication and assay parameters were optimized. The biosensor displays a linear response in the 0.5 to 50 ng·mL-1 IgG concentration range, and it has a 15 min assay time. The detection limit is 0.1 ng·mL-1 of IgG, which is 2.5 times lower than that when using a gold nanoparticle-based LFSB. In our perception, this assay has a wide potential for the detection of other proteins and species for which respective antibodies are available.

Gold Nanoparticle Coated Silica Nanorods for Sensitive Visual Detection of microRNA on a Lateral Flow Strip Biosensor.

We present a rapid and highly sensitive approach for visual detection of microRNA (miRNA) using a gold nanoparticles coated silica nanorod label and lateral flow strip biosensor. Gold nanoparticles were decorated on the silica nanorod surface by a seeding and growth procedure. A single strand DNA probe was immobilized on the gold nanoparticles-silica nanorod surface by a self-assembling process, and the formed DNA-gold nanoparticles-silica nanorod conjugate was used to construct the lateral flow nucleic acid biosensor for detecting miRNA. The captured gold nanoparticles-silica nanorods by sandwich-type hybridization reactions (DNA-RNA-DNA) on the test zone of the lateral flow nucleic acid biosensor produced the characteristic color bands, enabling visual detection of miRNA. After systematic optimization, the new lateral flow nucleic acid biosensor was capable of detecting 10 pM of the miRNA target without instrumentation, which is six times lower than that obtained with the gold nanoparticle-based lateral flow nucleic acid biosensor. The gold nanoparticles coated silica nanorod thus provides a new and sensitive nanolabel for visual detection of biological molecules on the lateral flow biosensor.

MWCNTs based high sensitive lateral flow strip biosensor for rapid determination of aqueous mercury ions

Here, we describe a disposable multi-walled carbon nanotubes (MWCNTs) labeled nucleic acid lateral flow strip biosensor for rapid and sensitive detection of aqueous mercury ions (Hg2+). Unlike the conventional colloidal gold nanoparticle based strip biosensors, the carboxylated MWCNTs were selected as the labeling substrate because of its high specific surface area for immobilization of recognition probes, improved stability and enhanced detection sensitivity of the strip biosensor. Combining the sandwich-type of T-Hg2+-T recognition mechanism with the optical properties of MWCNTs on lateral flow strip, optical black bands were observed on the lateral flow strips. Parameters (such as membrane category, the MWCNTs concentration, the amount of MWCNT-DNA probe, and the volume of the test probe) that govern the sensitivity and reproducibility of the sensor were optimized. The response of the optimized biosensor was highly linear over the range of 0.05–1ppb target Hg2+, and the detection threshold was estimated at 0.05 ppb within a 15-min assay time. The sensitivity was 10-fold higher than the conventional colloidal gold based strip biosensor. More importantly, the stability of the sensor was also greatly improved with the usage of MWCNTs as the labeling.