Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells.

Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.

Plasmonic Imaging of Multivalent NTD-Nucleic Acid Interactions for Broad-Spectrum Antiviral Drug Analysis.

The discovery and identification of broad-spectrum antiviral drugs are of great significance for blocking the spread of pathogenic viruses and corresponding variants of concern. Herein, we proposed a plasmonic imaging-based strategy for assessing the efficacy of potential broad-spectrum antiviral drugs targeting the N-terminal domain of a nucleocapsid protein (NTD) and nucleic acid (NA) interactions. With NTD and NA conjugated gold nanoparticles as core and satellite nanoprobes, respectively, we found that the multivalent binding interactions could drive the formation of core-satellite nanostructures with enhanced scattering brightness due to the plasmonic coupling effect. The core-satellite assembly can be suppressed in the presence of antiviral drugs targeting the NTD-NA interactions, allowing the drug efficacy analysis by detecting the dose-dependent changes in the scattering brightness by plasmonic imaging. By quantifying the changes in the scattering brightness of plasmonic nanoprobes, we uncovered that the constructed multivalent weak interactions displayed a 500-fold enhancement in affinity as compared with the monovalent NTD-NA interactions. We demonstrated the plasmonic imaging-based strategy for evaluating the efficacy of a potential broad-spectrum drug, PJ34, that can target the NTD-NA interactions, with the IC50 as 24.35 and 14.64 µM for SARS-CoV-2 and SARS-CoV, respectively. Moreover, we discovered that ceftazidime holds the potential as a candidate drug to inhibit the NTD-NA interactions with an IC50 of 22.08 µM from molecular docking and plasmonic imaging-based drug analysis. Finally, we validated that the potential antiviral drug, 5-benzyloxygramine, which can induce the abnormal dimerization of nucleocapsid proteins, is effective for SARS-CoV-2, but not effective against SARS-CoV. All these demonstrations indicated that the plasmonic imaging-based strategy is robust and can be used as a powerful strategy for the discovery and identification of broad-spectrum drugs targeting the evolutionarily conserved viral proteins.

Lanthanide Inorganic Nanoparticles Enhance Semiconducting Polymer Nanoparticles Afterglow Luminescence for In Vivo Afterglow/Magnetic Resonance Imaging.

Dual/multimodal imaging strategies are increasingly recognized for their potential to provide comprehensive diagnostic insights in cancer imaging by harnessing complementary data. This study presents an innovative probe that capitalizes on the synergistic benefits of afterglow luminescence and magnetic resonance imaging (MRI), effectively eliminating autofluorescence interference and delivering a superior signal-to-noise ratio. Additionally, it facilitates deep tissue penetration and enables noninvasive imaging. Despite the advantages, only a limited number of probes have demonstrated the capability to simultaneously enhance afterglow luminescence and achieve high-resolution MRI and afterglow imaging. Herein, we introduce a cutting-edge imaging platform based on semiconducting polymer nanoparticles (PFODBT) integrated with NaYF4@NaGdF4 (Y@Gd@PFO-SPNs), which can directly amplify afterglow luminescence and generate MRI and afterglow signals in tumor tissues. The proposed mechanism involves lanthanide nanoparticles producing singlet oxygen (1O2) upon white light irradiation, which subsequently oxidizes PFODBT, thereby intensifying afterglow luminescence. This innovative platform paves the way for the development of high signal-to-background ratio imaging modalities, promising noninvasive diagnostics for cancer.

Chemical Design of Activatable Photoacoustic Probes for Precise Biomedical Applications.

Photoacoustic (PA) imaging technology, a three-dimensional hybrid imaging modality that integrates the advantage of optical and acoustic imaging, has great application prospects in molecular imaging due to its high imaging depth and resolution. To endow PA imaging with the ability for real-time molecular visualization and precise biomedical diagnosis, numerous activatable molecular PA probes which can specifically alter their PA intensities upon reacting with the targets or biological events of interest have been developed. This review highlights the recent developments of activatable PA probes for precise biomedical applications including molecular detection of the biotargets and imaging of the biological events. First, the generation mechanism of PA signals will be given, followed by a brief introduction to contrast agents used for PA probe design. Then we will particularly summarize the general design principles for the alteration of PA signals and activatable strategies for developing precise PA probes. Furthermore, we will give a detailed discussion of activatable PA probes in molecular detection and biomedical imaging applications in living systems. At last, the current challenges and outlooks of future PA probes will be discussed. We hope that this review will stimulate new ideas to explore the potentials of activatable PA probes for precise biomedical applications in the future.

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

Zinc-Carnosine Metallodrug Network as Dual Metabolism Inhibitor Overcoming Metabolic Reprogramming for Efficient Cancer Therapy.

The targeting of tumor metabolism as a novel strategy for cancer therapy has attracted tremendous attention. Herein, we develop a dual metabolism inhibitor, Zn-carnosine metallodrug network nanoparticles (Zn-Car MNs), which exhibits good Cu-depletion and Cu-responsive drug release, causing potent inhibition of both OXPHOS and glycolysis. Notably, Zn-Car MNs can decrease the activity of cytochrome c oxidase and the content of NAD+, so as to reduce ATP production in cancer cells. Thereby, energy deprivation, together with the depolarized mitochondrial membrane potential and increased oxidative stress, results in apoptosis of cancer cells. In result, Zn-Car MNs exerted more efficient metabolism-targeted therapy than the classic copper chelator, tetrathiomolybdate (TM), in both breast cancer (sensitive to copper depletion) and colon cancer (less sensitive to copper depletion) models. The efficacy and therapy of Zn-Car MNs suggest the possibility to overcome the drug resistance caused by metabolic reprogramming in tumors and has potential clinical relevance.

Virus-like Plasmonic Nanoprobes for Quick Analysis of Antiviral Efficacy and Mutation-Induced Drug Resistance.

As the pathogenic viruses and the variants of concern greatly threaten human health and global public safety, the development of convenient and robust strategies enabling rapid analysis of antiviral drug efficacy and mutation-induced resistance is quite important to prevent the spread of human epidemics. Herein, we introduce a simple single-particle detection strategy for quick analysis of anti-infective drugs against SARS-CoV-2 and mutation-induced drug resistance, by using the wild-type and mutant spike protein-functionalized AuNPs as virus-like plasmonic nanoprobes. Both the wild-type and mutant virus-like plasmonic nanoprobes can form core-satellite nanoassemblies with the ACE2@AuNPs, providing the opportunity to detect the drug efficacy and mutation-induced resistance by detecting the changes of nanoassemblies upon drug treatment with dark-field microscopy. As a demonstration, we applied the single-particle detection strategy for quantitative determination of antiviral efficacy and mutation-induced resistance of ceftazidime and rhein. The mutations in the receptor-binding domain of Omicron variant could lead to an increase of EC50 values of ceftazidime and rhein, formerly from 49 and 57 µM against wild-type SARS-CoV-2, to 121 and 340 µM, respectively. The mutation-induced remarkable decline in the inhibitory efficacy of drugs was validated with molecule docking analysis and virus-like plasmonic nanoprobe-based cell-incubation assay. Due to the generality and feasibility of the strategy for the preparation of virus-like plasmonic nanoprobes and single-particle detection, we anticipated that this simple and robust method is promising for the discovery and efficacy evaluation of anti-infective drugs against different pathogenic viruses.

Vanin-1-Activated Chemiluminescent Probe: Help to Early Diagnosis of Acute Kidney Injury with High Signal-to-Noise Ratio through Urinalysis.

Acute kidney injury (AKI) is a common medical condition with high morbidity and mortality. Although urinalysis provides a noninvasive and convenient diagnostic method for AKI at the molecular level, the low sensitivity of current chemical probes used in urinalysis hinders the time diagnosis of AKI. Herein, we achieved the sensitive and early diagnosis of AKI by the development of a chemiluminescent probe CL-Pa suitable for detection of urinary Vanin-1. Vanin-1 is considered as an early and sensitive biomarker for AKI, while few chemical probes have been applied to for its efficient detection. By virtue of the low autofluorescence interference during urine imaging in the chemiluminescence model, CL-Pa could realize the monitoring of the up-regulated urinary Vanin-1 with a high signal-to-noise ratio (∼588). Importantly, under the help of CL-Pa, the up-regulation of urinary Vanin-1 of cisplatin-induced AKI mice at 12 h post cisplatin injection was detected, which was much earlier than clinical biomarkers (sCr and BUN) and change of kidney histology (48 h post cisplatin injection). Furthermore, using this probe, the fluctuation of urinary Vanin-1 of mice with different degrees of AKI was monitored. This study demonstrated the ability of CL-Pa in sensitively detecting drug-induced AKI through urinalysis and suggested the great potential of CL-Pa for early diagnosis of AKI and evaluate the efficiency of anti-AKI drugs clinically.

Organic Afterglow Nanoparticles in Bioapplications.

Organic afterglow nanoparticles are unique optical materials that emit light long after cessation of excitation. Due to their advantages of no need for real-time light excitation, avoiding autofluorescence, low imaging background, high signal-to-background ratio, deep tissue penetration, and high sensitivity, afterglow imaging technology has been widely used in cell tracking, biosensing, cancer diagnosis, and cancer therapy, which provides an effective technical method for the acquisition of molecular information with high sensitivity, specificity and real-time at the cellular and living level. In this review, we summarize and illustrate the recent progress of organic afterglow imaging, focusing on the mechanism of organic afterglow materials and their biological application. Furthermore, we also discuss the potential challenges and the further directions of this field.

Engineering of a novel D-A type fluorophore with hydrogen bond-induced enhanced emission property for sensitively detecting endogenous HOCl in living cells and tissues.

Fluorescence imaging has been widely employed for biomedical research and clinical diagnostics. With ease of synthesis and excellent photophysical properties, D-A type fluorophores are widely designed for fluorescence imaging. However, traditional D-A type fluorophores are solvatochromic which reduces the fluorescence brightness in the biological system. To solve this problem and build on our previous work, we devised a novel HIEE fluorophore MTC with typical anti-solvatochromic fluorescence. Furthermore, the activated fluorescent probe designed based on MTC showed excellent imaging performance. We believe that the strategy based on the fluorophores with typical anti-solvatohromic fluorescence can be a useful platform for designing fluorescent probes for high-brightness imaging in the biological system.

Molecular engineering of organic-based agents for in situ bioimaging and phototherapeutics.

In situ monitoring of the location and transportation of bioactive molecules is essential for deciphering diverse biological events in the field of biomedicine. In addition, obtaining the in situ information of lesions will provide a clear perspective for surgeons to perform precise resection in clinical surgery. Notably, delivering drugs or operating photodynamic therapy/photothermal therapy in situ by labeling the lesion regions of interest can improve treatment and reduce side effects in vivo. In various advanced imaging and therapy modalities, optical theranostic agents based on organic small molecules can be conveniently modified as needed and can be easily internalized into cells/lesions in a non-invasive manner, which are prerequisites for in situ bioimaging and precision treatment. In this tutorial review, we first summarize the in situ molecular immobilization strategies to retain small-molecule agents inside cells/lesions to prevent their diffusion in living organisms. Emphasis will be focused on introducing the application of these strategies for in situ imaging of biomolecules and precision treatment, particularly pertaining to why targeting therapy in situ is required.

Functional Xeno Nucleic Acids for Biomedical Application.

Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.

Progress and Perspective of Solid-State Organic Fluorophores for Biomedical Applications.

Fluorescent organic dyes have been extensively used as raw materials for the development of versatile imaging tools in the field of biomedicine. Particularly, the development of solid-state organic fluorophores (SSOFs) in the past 20 years has exhibited an upward trend. In recent years, studies on SSOFs have focused on the development of advanced tools, such as optical contrast agents and phototherapy agents, for biomedical applications. However, the practical application of these tools has been hindered owing to several limitations. Thus, in this Perspective, we have provided insights that could aid researchers to further develop these tools and overcome the limitations such as limited aqueous dispersibility, low biocompatibility, and uncontrolled emission. First, we described the inherent photophysical properties and fluorescence mechanisms of conventional, aggregation-induced emissive, and precipitating SSOFs with respect to their biomedical applications. Subsequently, we highlighted the recent development of functionalized SSOFs for bioimaging, biosensing, and theranostics. Finally, we elucidated the potential prospects and limitations of current SSOF-based tools associated with biomedical applications.

Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors.

Aminopeptidase N (APN) is capable of cleaving N-terminal amino acids from peptides with alanine in the N-terminal position and plays a key role in the growth, migration, and metastasis of cancer. However, reliable in situ information is hard to be obtained with the current APN-responsive molecular probes because the released fluorophores are cytoplasmic soluble and thus rapidly depart from the enzymatic reaction sites and spread out all over the cytoplasm. Here, we report a de novo precipitated fluorophore, HBPQ, which is completely insoluble in water and shows strong yellow solid emission when excited with a 405 nm laser. Owing to the controllable solid fluorescence of HBPQ by the protection-deprotection of phenolic hydroxyl, we further utilized HBPQ to design an APN-responsive fluorogenic probe (HBPQ-A) for the imaging of intracellular APN. Importantly, HBPQ-A can not only perform in situ imaging of APN in different organelles (e.g., lysosomes, mitochondria, endoplasmic reticula, and so forth) but also display a stable and indiffusible fluorescent signal for reliable mapping of the distribution of APN in living cells. In addition, through real-time imaging of APN in 4T1 tumors, we found that the fluorescent signal with high fidelity generated by HBPQ-A could remain constant even after 12 h, which further confirmed its diffusion-resistant ability and long-term reliable imaging ability. We believe that the precipitated fluorophore may have great potential for long-term in situ imaging.

Tumor-Specific Multipath Nucleic Acid Damages Strategy by Symbiosed Nanozyme@Enzyme with Synergistic Self-Cyclic Catalysis.

The high proliferation efficiency, redox imbalance, and elevated nucleic acid repair capabilities of tumor cells severely restrict the theranostic efficacy. Selectively interference chaotic tumors with devastating nucleic acid damages (NUDs) properties are expected to overcome theranostic barriers. Here, an exquisite catalytic-based strategy with comprehensive NUDs mechanisms is demonstrated. In this regard, enzyme (glucose oxidase, GOD) symbioses nanozyme Cu3+x (PO4 )2 through biomineralization (abbreviated as Cu@GOD), GOD can disorder the metabolism by consuming glucose, thereby inhibiting the nutrition supply for nucleic acid repair. GOD-catalyzed H2 O2 guarantees the self-cyclic glutathione depletion and reactive oxygen species generation caused by Cu3+x (PO4 )2 , resulted the reduced antioxidation defense and enhanced oxidation assault, ensures an indiscriminate NUDs ability. Moreover, the high photothermal effect of Cu3+x (PO4 )2 induces effective tumor inhibition. Consequently, this substantial multipath NUDs strategy, with potentials of suppressing the cytoprotective mechanisms, amplifying the cellular oxidative stress, and disrupting the redox balance to ensure substantial irreversible NUDs, completely breaks the obstacle of chaotic tumors, providing new conceptual thinking for tumor proliferation inhibition.

Smart Nanozyme Platform with Activity-Correlated Ratiometric Molecular Imaging for Predicting Therapeutic Effects.

Nanozymes with intrinsic enzyme-like characteristics have attracted enormous research interest in biological application. However, there is a lack of facile approach for evaluating the catalytic activity of nanozymes in living system. Herein, we develop a novel manganese-semiconducting polymer-based nanozyme (MSPN) with oxidase-like activity for reporting the catalytic activity of itself in acid-induced cancer therapy via ratiometric near-infrared fluorescence (NIRF)-photoacoustic (PA) molecular imaging. Notably, MSPN possess oxidase-like activity in tumor microenvironment, owing to the mixed-valent MnOx nanoparticles, which can effectively kill cancer cells. Because the semiconducting polymer (PFODBT) is conjugated with oxidase-responsive molecule (ORM), the catalytic activity of nanozyme can be correlated with the ratiometric signals of NIRF (FL695 /FL825 ) and PA (PA680 /PA780 ), which may provide new ideas for predicting anticancer efficacy of nanozymes in living system.

Learning from Artemisinin: Bioinspired Design of a Reaction-Based Fluorescent Probe for the Selective Sensing of Labile Heme in Complex Biosystems.

Labile heme (LH) is an important signaling molecule in virtually all organisms. However, specifically detecting LH remains an outstanding challenge. Herein, by learning from the bioactivation mechanism of artemisinin, we have developed the first LH-responsive small-molecule fluorescent probe, HNG, based on a 4-amino-1,8-naphthalimide (NG) fluorophore. HNG showed high selectivity for LH without interference from hemin, protein-interacting heme, and zinc protoporphyrin. Using HNG, the changes of LH levels in live cells were imaged, and a positive correlation of LH level with the degree of hemolysis was uncovered in hemolytic mice. Our study not only presents the first molecular probe for specific LH detection but also provides a strategy to construct probes with high specificity through a bioinspired approach.

Oxygen-Embedded Pentacene Based Near-Infrared Chemiluminescent Nanoprobe for Highly Selective and Sensitive Visualization of Peroxynitrite In Vivo.

Peroxynitrite (ONOO-) is involved in neurodegenerative, inflammatory, cardiovascular disorders, cancers, and other pathological progress. However, current imaging methods for sensing ONOO- usually suffer from high background/autofluorescence for fluorescent probes and poor selectivity/short emission wavelength for chemiluminescent probes. Herein, we present a novel chemiluminescent molecule (oxygen-embedded quinoidal pentacene) responsive to ONOO- for the first time, on the basis of which we rationally construct a near-infrared nanoprobe for detecting ONOO- via chemiluminescence resonance energy transfer (CRET) mechanism. Notably, our nanoprobe exhibits good selectivity, ultrahigh sensitivity (nanomole level), low background noise, fast response, and high water solubility. Moreover, the near-infrared emission from CRET offers higher tissue penetration of the chemiluminescent signal. Finally, our nanoprobe is further successfully applied to detecting endogenous ONOO- in mice with abdominal inflammation, drug-induced hepatotoxicity, or tumor models in vivo. In summary, the self-luminescing nanoprobes can act as an alternative visualizable tool for illuminating the mechanism of ONOO- involved in the specific pathological process.

Nitric Oxide-Activated "Dual-Key-One-Lock" Nanoprobe for in Vivo Molecular Imaging and High-Specificity Cancer Therapy.

Cancer treatments are confounded by severe toxic effects toward patients. To address these issues, activatable nanoprobes have been designed for specific imaging and destruction of cancer cells under the stimulation of specific cancer-associated biomarkers. Most activatable nanoprobes were usually activated by some single-factor stimulation, but this restricts therapeutic specificity between diseased and normal tissue; therefore, multifactor activation is highly desired. To this end, we herein develop a novel dual-stimuli responsive theranostic nanoprobe for simultaneously activatable cancer imaging and photothermal therapy under the coactivation of "dual-key" stimulation of "nitric oxide (NO)/acidity", so as to further improve the therapeutic specificity. Specifically, we have integrated a weak electron acceptor (benzo[c][1,2,5]thiadiazole-5,6-diamine) into a donor-π-acceptor-π-donor type chromophore. When the weak acceptor was oxidized by NO in acidic conditions to form a stronger acceptor (5H-[1,2,3]triazolo[4,5-f]-2,1,3-benzothiadiazole), the molecule absorption was significantly increased in the near-infrared region, based on the intramolecular charge transfer (ICT) mechanism. Under the dual-key stimulation of NO/acidity within the tumor associated with inflammation, the nanoprobe can correspondingly output dual signals for ratiometric photoacoustic and photothermal imaging of cancer in vivo and do so with enhanced accuracy and specificity. Our novel nanoprobe exhibited higher photoacoustic signal enhancement under dual-factor activation at 9.8 times that of NO and 132 times that of acidity alone, respectively. Moreover, through such dual activation of NO/acidity, the nanoprobe produces more differentiation of hyperthermia between tumor and normal tissues, to afford satisfactory photothermal therapy with minimal toxic side effects. Thus, our work presents a promising strategy for significantly improving the precision and specificity of cancer imaging and therapy.

Nanoscale Metal-Organic Framework Based Two-Photon Sensing Platform for Bioimaging in Live Tissue.

Nanoscale metal-organic frameworks (NMOFs) have been applied for biomedical sensing in recent years. However, it is still a great challenge to construct a highly efficient NMOFs fluorescent probe for sensing in a biological system, with high signal-to-noise ratio, photostability, and deep tissue penetration. Herein, for the first time, we report the two-photon metal-organic framework (TP-MOF) as a sensing platform. The design of TP-MOF is based on NMOFs incorporating a target-responsive two-photon organic moiety through click chemistry. PCN-58, as a model building block, was covalently modified with a small-molecule probe for H2S or Zn2+ as model analytes. TP-MOF probes retain the fluorescence-responsive properties of the TP organic moiety and possess excellent photostability and selectivity, as well as biocompatibility. Benefiting from the near-infrared (∼820 nm) excited two-photon fluorophore, TP-MOF probes serve to sense and image their respective targets in live cells and tissue slices with a penetration of 130 µm. The molecular design presented here bodes well for the extension to other MOFs displaying sensing components for other analytes of interest.