Portable dual-mode paper chips for highly sensitive and rapid determination of aflatoxin B1 via an aptamer-gated MOFs.

Paper chip as a representative microfluidic device has been mushroomed for rapid identification of contaminants in agro-food. However, the sensitivity and accuracy have still been challenged by inevitable background noise or interference in food matrix. Herein, we designed and fabricated a dual-mode paper chip (DPC) by assembling a patterned paper electrode with a platinum nanoparticles-treated colorimetric region through a flow channel. Dual-mode outputs were guided by an aptamer-gated UiO-66-NH2 metal-organic frameworks (MOFs). UiO-66-NH2 loaded with 3, 3', 5, 5'-tetramethylbenzidine (TMB) was controlled by a switch comprised of CdS quantum dots-aptamer. Aflatoxin B1 (AFB1, a kind of carcinogenic mycotoxin) target came and induced TMB release, triggering colorimetric and ECL signals on DPC, ultra-high sensitivity with a detection limit of 7.8 fg/mL was realized. The practicability of the DPC was also confirmed by spiking AFB1 in real corn samples. This portable paper-based device provides an ideal rapid detection platform tailored for diverse food contaminants analysis.

Structure modulation of two-dimensional transition metal chalcogenides: recent advances in methodology, mechanism and applications.

Together with the development of two-dimensional (2D) materials, transition metal dichalcogenides (TMDs) have become one of the most popular series of model materials for fundamental sciences and practical applications. Due to the ever-growing requirements of customization and multi-function, dozens of modulated structures have been introduced in TMDs. In this review, we present a systematic and comprehensive overview of the structure modulation of TMDs, including point, linear and out-of-plane structures, following and updating the conventional classification for silicon and related bulk semiconductors. In particular, we focus on the structural characteristics of modulated TMD structures and analyse the corresponding root causes. We also summarize the recent progress in modulating methods, mechanisms, properties and applications based on modulated TMD structures. Finally, we demonstrate challenges and prospects in the structure modulation of TMDs and forecast potential directions about what and how breakthroughs can be achieved.

High-performance electrochemiluminescence sensors based on ultra-stable perovskite quantum dots@ZIF-8 composites for aflatoxin B1 monitoring in corn samples.

Aflatoxin B1 (AFB1) that is prone to contaminate corns brings a serious threat to human health. Therefore, it is of great significance to construct novel detection methods for AFB1 tracing. Here, methylamine perovskite quantum dots (MP QDs) encapsulated by ZIF-8 metal-organic frameworks (MP QDs@ZIF-8) were prepared and then ultra-stable electrochemiluminescence (ECL) sensors were developed. By the confinement of cavities structure, multiple MP QDs were crystallized and embedded inside ZIF-8 to form MP QDs@ZIF-8, achieving stable and robust ECL responds in aqueous environment. Further combined with AFB1-imprinted polymer, the constructed ECL sensor showed good selectivity and ultra-sensitivity (the detection limit was 3.5 fg/mL, S/N = 3) with a wide linear range from 11.55 fg/mL to 20 ng/mL for AFB1 quantification. Satisfactory recoveries in corn samples indicated the reliable practicability of the proposed sensor for AFB1 assay. This work provided a novel pathway in designing high-performance ECL sensing platform for food safety.

Ruthenium(II) complex encapsulated multifunctional metal organic frameworks based electrochemiluminescence sensor for sensitive detection of hydrogen sulfide.

Metal organic frameworks (MOFs) based sensors exhibited a good deal of merits on sensitive detection like low reagent consumption, good chemical stability and high detection efficacy. Here, we reported a novel electrochemiluminescence (ECL) sensor which was based on tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)32+) encapsulated multifunctional metal organic frameworks (Ru-MOFs) as nanocarrier and nanoreactor for sensitive detection of H2S. Moreover, novel co-reactants NBD-amine was introduced into the ECL sensor as recognition probe. The introducing of Ru-MOFs successfully increased the amount of luminescent probe in the sensing system. At the same time, the Ru-MOFs as nanoreactors improved the molecule reaction efficiency inside the MOFs, including Ru(bpy)32+/co-reactants, and NBD-amine/H2S. Furthermore, the Ru-MOFs has superior adsorption capacity for H2S, which will facilitate the enrichment of H2S at the sensing interface. These were all contributed to enhance the ECL signal and improve the sensitivity of proposed ECL sensor. As a result, the proposed ECL sensor had excellent detection performance for H2S with the dynamic range from 1.0 × 10-11 mol L-1 to 1.0 × 10-4 mol L-1 and the detection limit was 2.5 × 10-12 mol L-1.

An ultrasensitive CH3NH3PbBr3 quantum dots@SiO2-based electrochemiluminescence sensing platform using an organic electrolyte for aflatoxin B1 detection in corn oil.

Mycotoxins contamination, especially aflatoxin B1 (AFB1) in edible oils, is a health hazard. Therefore, AFB1 trace analysis methods are urgently needed. Electrochemiluminescence (ECL) is a popular sensing method because of its low background interference and high sensitivity. However, existing ECL assays for AFB1 detection are based on aqueous rather than oil systems. Herein, we report a CH3NH3PbBr3 quantum dots (MAPB QDs)@SiO2-based ECL sensor for AFB1 quantification in corn oil using an organic electrolyte. The luminophore loading and stability of the MAPB QDs@SiO2 particles were significantly improved compared to those of bulky MAPB materials, resulting in an enhanced ECL response. Further, exploiting molecular imprinting technology, an ECL sensor for AFB1 detection with an ultra-low detection limit of 8.5 fg/mL was prepared. The reliability of the sensor was confirmed by comparable recoveries of corn oil samples with those obtained by high-performance liquid chromatography, indicating its potential for food safety evaluation.

Tetraphenylethylene-Functionalized Metal-Organic Frameworks with Strong Aggregation-Induced Electrochemiluminescence for Ultrasensitive Analysis through a Multiple Convertible Resonance Energy Transfer System.

Since aggregation-induced electrochemiluminescence (AIECL) combined the merits of aggregation-induced emission (AIE) and electrochemiluminescence (ECL), it has become a research hotspot recently. Herein, novel kinds of functional metal-organic frameworks (MOFs) with strong AIECL were reported through doping tetraphenylethylene (TPE) into UiO-66. Due to the porosity and highly ordered topological structure that caused the confinement effect of MOFs, the molecular motion of TPE was effectively limited within UiO-66, resulting in strong AIE. Meanwhile, the large specific surface area and porous structure of UiO-66 allowed TPE to react with coreactants more effectively, which was beneficial to ECL. Thus, the TPE-functionalized UiO-66 (TPE-UiO-66) showed excellent AIECL performance surprisingly. Inspired by this, a multiple convertible ECL resonance energy transfer (ECL-RET) system was constructed through a DNA Y structure that regulated the distance between the energy donor (TPE-UiO-66) and different energy acceptors (gold nanoparticles and Adriamycin). Furthermore, an ultrasensitive ECL biosensor for the detection of Mucin 1 (MUC1) was developed through the introduction of the novel ECL-RET system. In the presence of MUC1, the DNA Y structure was constructed, keeping the gold nanoparticles (AuNPs) away from TPE-UiO-66. Then, Adriamycin (Dox) could be embedded in the DNA Y structure and act as an energy acceptor to receive the energy of TPE-UiO-66, which made the biosensor produce a strong ECL response. As expected, the developed ECL biosensor exhibited superior detection performance for MUC1. This work provided a novel way to realize AIECL and board the application of AIECL in analytical chemistry.

A near-infrared light-controlled, ultrasensitive one-step photoelectrochemical detection of dual cell apoptosis indicators in living cancer cells.

Here, a near-infrared (NIR) light-controlled, ultrasensitive one-step photoelectrochemical (PEC) strategy was constructed to simultaneously detect cell apoptosis indicators, phosphatidylserine (Pho) and sodium-potassium adenosine triphosphatase (Sat), on living cancer cells. Using NIR light as excitation, the signal probe methylene blue (Tagkinetic) could be released, leading to a gradually decreased photocurrent signal Ikinetic; meanwhile, the photocurrent Istable of the signal probe carbon quantum dots (Tagstable) remained stable. The simultaneous detection of Pho and Sat could be achieved based on rapid one-step PEC detection under single NIR light with the assistance of a smart signal decryption strategy with Ikinetic and Istable. Importantly, this proposal provides more effective drug candidates with milder pharmaceutical effect but improved safety.

Iron doped graphitic carbon nitride with peroxidase like activity for colorimetric detection of sarcosine and hydrogen peroxide.

The successful synthesis is reported of Mn, Fe, Co, Ni, Cu-doped g-C3N4 nanoflakes via a simple one-step pyrolysis method, respectively. Among them, the Fe-doped g-C3N4 nanoflakes exhibited the highest peroxidase-like activity, which can be used for colorimetric detection of hydrogen peroxide (H2O2) and sarcosine (SA), within the detection ranges of 2-100 µM and 10-500 µM and detection limits of 1.8 µM and 8.6 µM, respectively. The catalytic mechanism of the Fe-doped g-C3N4 nanoflake was also explored and verified the generation of hydroxyl radical (•OH) through fluorescence method. It is believed that the Fe-doped g-C3N4 nanoflakes as enzyme mimics will greatly promote the practical applications in a variety of fields in the future including biomedical science, environmental governance, antibacterial agent, and bioimaging due to their extraordinary catalytic performance and stability. Graphical abstract.

A synergistic approach to enhance the photoelectrochemical performance of carbon dots for molecular imprinting sensors.

Nanoscale carbon dots (CDs) have drawn increasing attention in photoelectrochemical (PEC) sensors for biotoxin detection owing to their many merits including excellent optical, electric and photoelectric properties. In this work, a novel strategy is proposed to improve the photoelectrical response performance of CDs by taking advantage of the synergistic effect of nitrogen and sulfur co-doping and copper phthalocyanine non-covalent functionalization approaches, which rightly adjusts the energy level of CDs, optimization of intimate interfacial contact, extension of the light absorption range, and enhancement of charge-transfer efficiency. This work demonstrates that heteroatom doping and chemical functionalization can endow CDs with various new and improved physicochemical, optical, and structural performances. This synergy contributes enormously to the molecular imprinting photoelectrochemical (MIP-PEC) sensor for toxin detection, and the work typically provided a wide linear range of 0.01 to 1000 ng mL-1 with a detection limit of 0.51 pg mL-1 for ochratoxin A (OTA).

Highly stable Ru-complex-grafted 2D metal-organic layer with superior electrochemiluminescent efficiency as a sensing platform for simple and ultrasensitive detection of mucin 1.

This work utilized ultrathin metal-organic layer (MOL) to immobilize luminophores for effectively shortening the ion/electron-transport distance and relieving the diffusional constraints of ion/electron, which greatly enhanced the ECL efficiency and intensity. Moreover, the MOL's immobilization amount of luminophores should be higher than these of bulk MOFs because MOLs possess more accessible postmodification sites for the luminophores with minimal diffusion barriers. As expected, our proof-of-concept experiment indicated that the Hf-MOL's loading number of Ru(bpy)2(mcpbpy)2+ was about 1.74 times that of a 3D mesoporous MOF (PCN-777), and the ECL efficiency and intensity of PEI@Ru-Hf-MOL were around 1.27 times and 14.5 times those of PEI@Ru-PCN-777, respectively. In view of these merits, this work utilized the prepared PEI@Ru-Hf-MOL as a highly efficient sensing platform for simple, rapid and sensitive detection of mucin 1, which exhibited a broad linearity from 1 fg/mL to 10 ng/mL and a low detection limit of 0.48 fg/mL. This work provided a practicable strategy to develop high-performance ECL materials, and therefore opened up a new avenue to design ultrasensitive ECL biosensors, which expanded the application potential of MOLs in ECL assays.

Enhanced electrochemiluminescence of gold nanoclusters via silver doping and their application for ultrasensitive detection of dopamine.

Gold nanoclusters (Au NCs) are a type of emerging ECL emitter with molecule-like properties and low toxicity that hold great potential for sensing application. However, the application of Au NCs in ECL sensing is still limited due to their low ECL efficiency. In this work, we provided an effective way to enhance the ECL efficiency of Au NCs. By doping Ag on GSH-protected Au NCs to form bimetallic clusters (GSH-Ag/Au NCs), the ECL efficiency of Au NCs was greatly improved. Based on the enhanced ECL signal of Au NCs, an ultrasensitive ECL sensor was constructed for the detection of dopamine (DA). DA exhibited a prominent ECL quenching effect towards the formed bimetallic GSH-Ag/Au NCs which can be used for DA detection. The proposed ECL sensor exhibited excellent sensitivity, selectivity and stability and had a wide linear range from 10 nM to 1 mM with a low detection limit of 2.3 nM (S/N = 3). More importantly, this work provided a potential method to improve the ECL properties of Au NCs and widen their analytical application.

An ATP-fueled nucleic acid signal amplification strategy for highly sensitive microRNA detection.

Herein, an adenosine triphosphate (ATP)-fueled nucleic acid signal amplification strategy based on toehold-mediated strand displacement (TMSD) and fluorescence resonance energy transfer (FRET) was proposed for highly sensitive detection of microRNA-21. More importantly, the target microRNA-21 could be regenerated with ATP as the fuel rather than a nucleotide segment in conventional approaches, which made the proposed strategy simple and efficient due to the high affinity and strength of the aptamer-target interaction.

Highly sensitive electrochemiluminescence immunosensor based on ABEI/H2O2 system with PFO dots as enhancer for detection of kidney injury molecule-1.

In this work, poly[9,9-dioctylfluorenyl-2,7-diyl] (PFO) dots is discovered to display an appealing dual enhancement effect for the electrochemiluminescence (ECL) system of N-(aminobutyl)-N-(ethylisoluminol)/hydrogen peroxide (ABEI/H2O2), which not only enhances the ECL intensity of ABEI but also catalyzes decomposition of H2O2 to further amplify the ECL signal of ABEI. Owing to the electronegative property of PFO dots, electropositive ABEI-PEI as ECL reagent could be adsorbed on their surface and thus form a novel luminescence emitter (ABEI-PEI-PFO dots) with high ECL efficiency based on electrostatic attraction. Meanwhile, the water solubility and stability of this emitter are improved in virtue of the amine-rich property of ECL reagent (ABEI-PEI), which could increase the luminous efficiency of ECL reaction in aqueous solution. To increase the electron transfer efficiency, Pt nanoparticles (PtNPs) supported on reduced graphene oxide nanosheets (RGOs) via a onepot synthetic strategy are chosen as immobilizing platform for the ECL emitter (ABEI-PEI-PFO dots). Herein, the obtained dual-amplifed ABEI-PEI-PFO dots-RGOs/PtNPs complex is served as an ideal nanocarrier to capture detection antibody (Ab2). According to sandwiched immunoreaction, a highly sensitive ECL immunosensor is constructed for the detection of kidney injury molecule-1 (KIM-1) with a linearity from 50 fg mL-1 to 1 ng mL-1 and a detection limit of 16.7 fg mL-1. The developed ECL emitter combining dual amplified property for signal enhancement purpose would provide new thought and potential for sensitive bioanalysis and clinical application.

Ultrasensitive Fluorescent Assay Based on a Rolling-Circle-Amplification-Assisted Multisite-Strand-Displacement-Reaction Signal-Amplification Strategy.

Heavy metal ions are persistent environmental contaminants and pose a great threat to human health, which has prompted demand for new methods to selectively identify and detect these metal ions. Herein, a novel fluorescent assay based on a rolling-circle-amplification (RCA)-assisted multisite-strand-displacement-reaction (SDR) signal-amplification strategy was proposed for the ultrasensitive detection of heavy metal ions with lead ions (Pb2+) as a model. The proposed strategy not only achieved the target recycling but also introduced RCA induced by released DNAzyme. Most importantly, the RCA product was adapted as the initiator to provide multiple sites for SDR, which could displace signal duplexes from RCA products to effectively avoid the self-quenching of signal-probe assembly on the RCA product. Therefore, the amplification efficiency and the detection sensitivity could be improved significantly. As expected, the proposed strategy demonstrated good performance for the determination of Pb2+ with a linear range from 0.1 to 50 nM and a detection limit down to 0.03 nM. Using this strategy for intracellular-Pb2+ detection, a favorable property was obtained. Furthermore, the proposed strategy could be also expanded for the determination of microRNA, proteins, and other biomolecules, offering a novel avenue for environmental assays and clinical diagnostics.

Highly Stable Mesoporous Luminescence-Functionalized MOF with Excellent Electrochemiluminescence Property for Ultrasensitive Immunosensor Construction.

In this work, a novel mesoporous luminescence-functionalized metal-organic framework (Ru-PCN-777) with high stability and excellent electrochemiluminescence (ECL) performance was synthesized by immobilizing Ru(bpy)2(mcpbpy)2+ on the Zr6 cluster of PCN-777 via a strong coordination bond between Zr4+ and -COO-. Consequently, the Ru(bpy)2(mcpbpy)2+ could not only cover the surface of PCN-777 but also graft into the interior of PCN-777, which greatly increased the loading amount of Ru(bpy)2(mcpbpy)2+ and effectively prevented the leaching of the Ru(bpy)2(mcpbpy)2+ resulting in a stable and high ECL response. Considering the above merits, we utilized the mesoporous Ru-PCN-777 to construct an ECL immunosensor to detect mucin 1 (MUC1) based on proximity-induced intramolecular DNA strand displacement (PiDSD). The ECL signal was further enhanced by the enzyme-assisted DNA recycling amplification strategy. As expected, the immunosensor had excellent sensitivity, specificity, and responded wide linearly to the concentration of MUC1 from 100 fg/mL to 100 ng/mL with a low detection limit of 33.3 fg/mL (S/N = 3). It is the first time that mesoporous Zr-MOF was introduced into ECL system to assay biomolecules, which might expand the application of mesoporous metal-organic frameworks (MOFs) in bioanalysis. This work indicates that the use of highly stable mesoporous luminescence-functionalized MOFs to enhance the ECL intensity and stability is a feasible strategy for designing and constructing high-performance ECL materials, and therefore may shed light on new ways to develop highly sensitive and selective ECL sensors.

Electrochemiluminescent Pb2+-Driven Circular Etching Sensor Coupled to a DNA Micronet-Carrier.

Herein, an ultrasensitive electrochemiluminescent (ECL) strategy was designed based on the fabrication of a multi-interface DNA micronet-carrier via layer by layer hybridization of double-stranded DNAzyme-substrate to immobilize large amounts of ECL indicator, [Ru(dcbpy)2dppz]2+, in double-strand DNA on the electrode surface, generating enhanced ECL signals. When the double-stranded structures were cleaved circularly via Pb2+ in the detection sample, the ECL indicator was released, which resulted in a decreased ECL signal associated with the concentration of Pb2+, that had higher sensitivity and wider linear range. As a result, the developed ECL strategy exhibited a linear range from 50 pM to 500 µM with a detection limit of 4.73 pM, providing an alternative analytical strategy with excellent properties, including a high sensitivity and a wide linear range. Importantly, the ECL strategy could be readily expanded for various metal ions, proteins, nucleotide sequences, and cells, offering a simple and efficient technology for both environmentally safe assays and clinical diagnostics.

Intramolecular Self-Enhanced Nanochains Functionalized by an Electrochemiluminescence Reagent and Its Immunosensing Application for the Detection of Urinary ß2-Microglobulin.

In this study, polyethylenimine (PEI) is discovered to possess a noticeable amplification effect for the electrochemiluminescence (ECL) of N-(aminobutyl)-N-(ethylisoluminol) (ABEI); thus, a novel self-enhanced ECL reagent (ABEI-PEI) is prepared by covalent cross-linking. Because of the shortened electron-transfer path and reduced energy loss, the intramolecular ECL reaction between ABEI and PEI exhibited enhanced luminous efficiency compared with the traditional intermolecular ECL reaction. Owing to the amine-rich property of PEI, abundant ABEI could be immobilized on the molecular chains of PEI to strengthen the luminous intensity of ABEI-PEI. On account of the reducibility of remaining amino groups, ABEI-PEI, as the self-enhanced ECL reagent, has also been chosen as a reductant and stabilizer for in situ preparation of Au@Ag nanochains (Au@AgNCs) which has the catalytic activity for the ECL reaction. Moreover, using ABEI-PEI as a template to directly prepare Au@AgNCs realizes the immobilization of the ECL reagent with large amounts. Meanwhile, in virtue of the electropositivity of ABEI-PEI-capped Au@AgNCs (ABEI-PEI-Au@AgNCs), polyacrylic acid (PAA) with electronegativity is pervaded on the surface of nanochains and further chelates with Co2+ to form an ABEI-PEI-Au@AgNCs-PAA/Co2+ complex, which could introduce Co2+ as a catalyst to promote H2O2 decomposition and thus oxidize ABEI to produce an enhanced ECL signal. Here, the obtained self-enhanced ABEI-PEI-Au@AgNCs-PAA/Co2+ complex is utilized to capture the detection antibody (Ab2). According to sandwiched immunoreactions, a sensitive ECL immunosensor is constructed for the detection of ß2-microglobulin with a wide linearity from 0.01 pg mL-1 to 200 ng mL-1 and a detection limit of 3.3 fg mL-1.

Universal Ratiometric Photoelectrochemical Bioassay with Target-Nucleotide Transduction-Amplification and Electron-Transfer Tunneling Distance Regulation Strategies for Ultrasensitive Determination of microRNA in Cells.

A universal ratiometric photoelectrochemical (PEC) bioassay, which could be readily expanded for ultrasensitive determination of various targets in complex biological matrixes, was established by coupling a target-nucleotide transduction-amplification with DNA nanomachine mediated electron-transfer tunneling distance regulation strategies. With the help of target-nucleotide transduction-amplification strategy, the one input target signal could be transducted to corresponding multiple output DNA signals by nucleotide specific recognition technology, simultaneously leading to an efficient signal amplification for target. Then the output DNA could initiate the formation of four-way junction DNA nanomachine through binding-induced combination, by which the electron-transfer tunneling distance between photoactive materials and sensing interface could be regulated, simultaneously resulting an enhanced photocurrent signal from SiO2@methylene blue (SiO2@MB) as wavelength-selective photoactive material in close proximity to sensing interface and a reduced photocurrent signal from another wavelength-selective photoactive material CdS quantum dots (CdS QDs) away from sensing interface for photocurrent signal ratio calculation. Using microRNA-141 (miRNA-141) as target model, the constructed biosensor demonstrated favorable accuracy and excellent sensitivity down to the femtomolar level. Impressively, the proposed assay overcame the heavy dependence of target on photoactive materials in current ratiometric PEC assay and demonstrated admirably universal applicability for determination of various targets such as metal ions, miRNAs, DNAs, and proteins by merely two different photoactive materials (SiO2@MB and CdS QDs), paving the way to application of universal ratiometric PEC assay in environmental tests, clinical diagnosis, and other related subjects.

An efficient target-intermediate recycling amplification strategy for ultrasensitive fluorescence assay of intracellular lead ions.

An ultrasensitive fluorescence assay for intracellular Pb2+ determination was proposed through target-intermediate recycling amplification based on metal-assisted DNAzyme catalysis and strand displacement reactions. Compared with only target recycling-based fluorescence assay with an M amplification ratio, the proposed assay could achieve an M × N amplification ratio to obtain an improved sensitivity by more than 10 times, in which M and N are the amplification ratios of target recycling and intermediate recycling, respectively. Remarkably, this proposed ultrasensitive fluorescence assay could be applied to the determination of various analytes with the well-designed detection probe, especially in intracellular assay, providing a promising tool for clinical diagnosis and biomedical detection.

A sensitive immunosensor via in situ enzymatically generating efficient quencher for electrochemiluminescence of iridium complexes doped SiO2 nanoparticles.

A sensitive electrochemiluminescent (ECL) sandwich immunosensor was proposed herein based on the tris (2-phenylpyridine) iridium [Ir(ppy)3] doped silica nanoparticles (SiO2@Ir) with improved ECL emission as signal probes and glucose oxidase (GOD)-based in situ enzymatic reaction to generate H2O2 for efficiently quenching the ECL emission of SiO2@Ir. Typically, the SiO2@Ir not only increased the loading amount of Ir(ppy)3 as ECL indicators with high ECL emission, but also improved their water-solubility, which efficiently enhanced the ECL emission. Furthermore, by the efficient quench effect of H2O2 from in situ glucose oxidase (GOD)-based enzymatic reaction on the ECL emission of SiO2@Ir, a signal-off ECL immunsensor could be established for sensitive assay. With N-terminal of the prohormone brain natriuretic peptide (BNPT) as a model, the proposed ECL assay performed high sensitivity and low detection limit. Importantly, the proposed sensitive ECL strategy was not only suitable for the detection of BNPT for acute myocardial infarction, but also revealed a new avenue for early diagnosis of various diseases via proteins, nucleotide sequence, microRNA and cells.