RESUMEN
Sensitive detection of extracellular vesicles (EVs) as emerging biomarkers has shown great promises for disease diagnosis. Plasmonic metal nanostructures conjugated with molecules that bind specific biomarker targets are widely used for EVs sensing but involve tradeoffs between particle-size-dependent signal intensity and conjugation efficiency. One solution to this problem would be to induce nucleation on nanoparticles that have successfully bound a target biomarker to permit in situ nanoparticle growth for signal amplification, but approaches that are evaluated to date require harsh conditions or lack nucleation specificity, prohibiting their effective use with most biological specimens. This study describes a one-step in situ strategy to induce monocrystalline copper shell growth on gold nanorod probes without decreasing signal by disrupting probe-target interactions or lipid bilayer integrity to enable EV biomarker detections. This approach increases the detected nanoparticle signal about two orders of magnitude after a 10 min copper nanoshell growth reaction. This has significant implications for improved disease detection, as indicated by the ability of a novel immunoassay using this approach to detect low abundance EVs carrying a pathogen-derived biomarker, after their direct capture from serum, to facilitate the diagnosis of tuberculosis cases in a diagnostically challenging pediatric cohort.
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Vesículas Extracelulares , Nanopartículas , Humanos , Niño , Cobre/metabolismo , Biomarcadores/análisis , Membrana Dobles de Lípidos/metabolismo , Vesículas Extracelulares/metabolismoRESUMEN
BACKGROUND: Novel approaches that allow early diagnosis and treatment monitoring of both human immunodeficiency virus-1 (HIV-1) and tuberculosis disease (TB) are essential to improve patient outcomes. METHODS: We developed and validated an immuno-affinity liquid chromatography-tandem mass spectrometry (ILM) assay that simultaneously quantifies single peptides derived from HIV-1 p24 and Mycobacterium tuberculosis (Mtb) 10-kDa culture filtrate protein (CFP10) in trypsin-digested serum derived from cryopreserved serum archives of cohorts of adults and children with/without HIV and TB. RESULTS: ILM p24 and CFP10 results demonstrated good intra-laboratory precision and accuracy, with recovery values of 96.7% to 104.6% and 88.2% to 111.0%, total within-laboratory precision (CV) values of 5.68% to 13.25% and 10.36% to 14.92%, and good linearity (r2 > 0.99) from 1.0 to 256.0â pmol/L and 0.016 to 16.000â pmol/L, respectively. In cohorts of adults (n = 34) and children (n = 17) with HIV and/or TB, ILM detected p24 and CFP10 demonstrated 85.7% to 88.9% and 88.9% to 100.0% diagnostic sensitivity for HIV-1 and TB, with 100% specificity for both, and detected HIV-1 infection earlier than 3 commercial p24 antigen/antibody immunoassays. Finally, p24 and CFP10 values measured in longitudinal serum samples from children with HIV-1 and TB distinguished individuals who responded to TB treatment from those who failed to respond or were untreated, and who developed TB immune reconstitution inflammatory syndrome. CONCLUSIONS: Simultaneous ILM evaluation of p24 and CFP10 results may allow for early TB and HIV detection and provide valuable information on treatment response to facilitate integration of TB and HIV diagnosis and management.
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Infecciones por VIH , VIH-1 , Mycobacterium tuberculosis , Adulto , Niño , Humanos , Espectrometría de Masas en Tándem , Infecciones por VIH/diagnóstico , Péptidos , Cromatografía Liquida , Sensibilidad y EspecificidadRESUMEN
One major frustration in developing antibiotics is that bacteria can quickly develop resistance that would require an entirely new cycle of research and clinical testing to overcome. Although plenty of bactericidal nanomaterials have been developed against increasingly severe superbugs, few reports have studied the resistance to these nanomaterials. Herein, we show that antibacterial 4,6-diamino-2-pyrimidine thiol (DAPT)-capped gold nanoparticles (AuDAPTs) can induce a 16-fold increased minimum inhibitory concentration (MIC) of E. coli only after very long term exposure (183 days), without developing cross-resistance to commercialized antibiotics. Strikingly, we recovered the bactericidal activities of AuDAPTs to the resistant strain by tuning the sizes of AuDAPTs without employing new chemicals. Such slow, easy-to-handle resistance induced by AuDAPTs is unprecedented compared to traditional antibiotics or other nanomaterials. In addition to the novel antibacterial activities and biocompatibilities, our approach will accelerate the development of gold nanomaterial-based therapeutics against multi-drug-resistant (MDR) bacterial infections.
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Infecciones Bacterianas , Nanopartículas del Metal , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Escherichia coli , Oro , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Conventional transverse relaxation time (T2)-mediated magnetic resonance sensors (MRS) that utilizing the target-induces state change of magnetic nanoparticles (MNPs) mainly suffer from low sensitivity. Recent T2-MRS that based on target-induced amount change of MNPs can achieve a higher sensitivity, but these sensors can hardly accommodate small molecules. We herein develop an ultrasensitive T2-MRS that enable the detection of small molecules based on cascade bioorthogonal reactions (BRs)-realized MNPs binding and assembly. Benefiting from rapid and highly selective cascade BRs, a single small molecule target can not only increase MNPs binding but also assembly MNPs, which greatly amplifies T2 signal for sensing based on both the state and amount change of MNPs for the first time. Our strategy is capable of sensing chlorpyrifos with a liner range of 0.1 ng/mL to 1000 ng/mL. We justify the practicability of our assay by detecting chlorpyrifos in apple and cabbage samples, whose accuracy is higher than that of enzyme linked immunosorbent assay. Our assay provides a cascade BRs-mediated MRS that can greatly broaden the use of T2-based MRS for ultrasensitive sensing trace small molecules in complex samples.
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Brassica/química , Cloropirifos/análisis , Nanopartículas de Magnetita/química , Malus/química , Bibliotecas de Moléculas Pequeñas/análisis , Sitios de Unión , Espectroscopía de Resonancia MagnéticaRESUMEN
We have developed an alginate hydrogel-embedded capillary sensor (AHCS) for naked eye-based quantification of immunoassay. Alkaline phosphatase (ALP) can modulate gel-sol transformation to increase the permeability of Cu2+-cross-linked alginate hydrogel film in the AHCS, followed by solution exchange into the capillary. Through measuring the length of the liquid phase of the microfluidics in the capillary at a given time, the concentration of the ALP could be quantified with the naked eye. Since ALP is widely applied as a signal reporter for immunoassays, the AHCS could easily accommodate conventional immune sensing platforms. We justify the practicality of AHCS with hepatitis B virus surface antigen (HBsAg) in serum samples and got comparable results with commercialized immunoassay. This AHCS is easy to make and use, effective in cost, and robust in quantification with the naked eye, showing great promise for next generation point-of-care testing.
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Alginatos , Antígenos de Superficie de la Hepatitis B/análisis , Hidrogeles , Inmunoensayo/métodos , Fosfatasa Alcalina/química , Antígenos de Superficie de la Hepatitis B/sangre , HumanosRESUMEN
Conventional magnetic sensors usually employ Fe-based magnetic materials as signal probes. In this work, we find that Cu(II) is also a useful longitudinal relaxation time (T1) signal-based magnetic probe. We adopt bathocuproinedisulfonic acid disodium salt hydrate (BCS) to chelate Cu(I) and form a stable Cu(I)-BCS complex in aqueous solution and find the significant difference in the T1 value of water protons between Cu(II) aqueous solution and Cu(I)-BCS complex aqueous solution. Redox reaction can convert Cu(II) to Cu(I) followed by the complexation of BCS, which results in apparent change of T1 that can serve as magnetic signal readout, which is the basis of this Cu-T1 sensor. Many redox reactions between Cu(II) and Cu(I) allow this Cu-T1 sensor to not only realize "one-step mode" assay such as ascorbic acid, protein, and alkaline phosphatase but also enable "multi-step mode" immunoassay, such as biomacromolecules and small molecules. This Cu-T1 sensor employs Cu ion as signal readout, providing an alternative tool for biochemical analysis.
RESUMEN
Conventional magnetic relaxation switching (MRS) sensor suffers from its relatively low sensitivity when it comes to the analysis of trace small molecules in complicated samples. To meet this challenge, we develop a cascade reaction-mediated magnetic relaxation switching (CR-MRS) sensor, based on the assembly of silver nanoparticles (Ag NPs) and magnetic nanoparticles (MNPs) to improve the sensitivity of conventional MRS. The cascade reaction triggered by alkaline phosphatase generates ascorbic acid, which reduces Ag+ to Ag NPs that can assemble the initially dispersed MNPs to form magnetic/silver nanoassemblies, thus modulating the state of MNPs to result in the change of transverse relaxation time. The formed magnetic/silver nanoassemblies can greatly enhance the state change of MNPs (from dispersed to aggregated) and dramatically improve the sensitivity of traditional MRS sensor, which makes this CR-MRS sensor a promising platform for highly sensitive detection of small molecules in complicated samples.
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Técnicas Biosensibles , Cloranfenicol/análisis , Nanopartículas de Magnetita/química , Leche/química , Plata/química , Animales , Fenómenos Magnéticos , Estructura MolecularRESUMEN
The main challenge of paramagnetic ions-mediated magnetic sensors is their relatively low sensitivity. In this study, we observe the amplification of longitudinal relaxation time (T1) signal when Fe2+ transforms into Fe3+ followed by the coordination of potassium thiocyanate (KSCN) and develop a sensitive Fe-T1 sensor based on the coordination chemistry between KSCN and Fe3+ to amplify the T1 signal for detecting a series of targets, such as hydrogen peroxide, glucose, and antigen/antibody. We justify the practicability of our assay by successfully detecting tetracycline in milk samples and hepatitis C virus in clinical samples with satisfactory accuracy. This KSCN-mediated Fe-T1 sensor not only realizes biochemical analysis and immunoassay with higher sensitivity but also retains many advantages of paramagnetic ions-mediated magnetic sensors (good stability and straightforward operation), which holds great promise for the detection of a range of targets of interest in complex samples.
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Técnicas Biosensibles/métodos , Cloruros/química , Compuestos Férricos/química , Compuestos Ferrosos/química , Tiocianatos/química , Animales , Complejos de Coordinación/química , Glucosa/análisis , Hepacivirus/aislamiento & purificación , Hepatitis C/virología , Humanos , Peróxido de Hidrógeno/análisis , Inmunoensayo/métodos , Leche/química , Tetraciclina/análisisRESUMEN
We develop a convenient, colorimetric assay (Au/PEI) for rapid iodide (I-) determination that can be prepared facilely by mixing commercially available chemicals including tetrachloroauric acid (HAuCl4) and poly(ether imide) (PEI), and the assay can be carried out directly by adding the samples to the assay without any pretreatment and additional procedure. Au/PEI operates on the principle that I- accelerates the formation of Au NPs, which leads to a visible color change from light yellow to red for naked-eye readout with high specificity. We integrate our assay on solid devices including gel hybrids (Au/PEI/GH) and filter paper (Au/PEI paper) to satisfy the demand of point-of-care testing and justify the practicality by detecting I- in lake water that was supplemented with 10, 20, or 40 µM of I-. Au/PEI/GH with the limit of detection of 0.35 µM can satisfy the detection of drinking water based on the guidelines (1.2 µM) set by the Chinese government, and Au/PEI paper can be used even after 1 year of storage. Such assays provide a convenient and straightforward choice for routine, on-site I- tests.
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Colorimetría/métodos , Yoduros/análisis , Polímeros/química , Oro/química , Lagos/química , Límite de Detección , Nanopartículas del Metal/química , Factores de Tiempo , Agua/químicaRESUMEN
Current magnetic relaxation switching (MRS) sensors for detection of trace targets in complex samples still suffer from limitations in terms of relatively low sensitivity and poor stability. To meet this challenge, we develop a longitudinal relaxation time (T1)-based nanosensor by using Mn2+ released from the reduction of a MnO2 nanoassembly that can induce the change of T1, and thus can greatly improve the sensitivity and overcome the "hook effect" of conventional MRS. Through the specific interaction between antigen and the antibody-functionalized MnO2 nanoassembly, the T1 signal of Mn2+ released from the nanoassembly is quantitatively determined by the antigen, which allows for highly sensitive and straightforward detection of targets. This approach broadens the applicability of magnetic biosensors and has great potential for applications in early diagnosis of disease biomarkers.
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Técnicas Biosensibles , Inmunoensayo , Compuestos de Manganeso/análisis , Manganeso/análisis , Nanopartículas/análisis , Óxidos/análisis , Humanos , Estructura Molecular , Oxidación-Reducción , Tamaño de la Partícula , Propiedades de Superficie , Factores de TiempoRESUMEN
The colorimetric immunoassay based on gold nanoparticles (AuNPs) can hardly enable simultaneous detection of multiple biomarkers in vastly different concentrations (e.g., pg/mL-µg/mL) because of its narrow dynamic range. In this work, we demonstrate an immunoassay with tunable detection range by using peptide-mediated controlled aggregation of surface modification-free AuNPs. Alkaline phosphatase (ALP) removes the phosphate group of the peptide to yield a positively charged product, which triggers the aggregation of negatively charged AuNPs and the color change of the AuNPs solution from red to blue with naked-eye readout. We design and screen 20 kinds of phosphorylated peptides to obtain a broad and controllable detection range for ALP sensing and apply them for detecting multiple inflammatory biomarkers in clinical samples. Our assay realizes straightforward, multiplexed, and simultaneous detection of multiple clinical biomarkers with tunable detection range (from pg/mL to µg/mL) in the same run and holds great potential for chemical/biochemical analysis.
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Oro/química , Inmunoensayo/métodos , Nanopartículas del Metal/química , Péptidos/química , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Proteína C-Reactiva/análisis , Colorimetría , Humanos , Interleucina-6/sangre , Límite de Detección , Modelos Moleculares , Péptidos/metabolismo , Polipéptido alfa Relacionado con Calcitonina/sangre , Conformación ProteicaRESUMEN
Chemical regulation of enzyme-mimic activity of nanomaterials is challenging because it requires a precise understanding of the surface chemistry and mechanism, and rationally designed applications. Herein, Ag+ -gated peroxidase activity is demonstrated by successfully modulating surface chemistry of cetyltrimethylammonium bromide-capped gold nanoparticles (CTAB-AuNPs). A surface blocking effect of long-chain molecules on surfaces of AuNPs that inhibit peroxidase activity of AuNPs is found. Ag+ ions can selectively bind on the surfaces of AuNPs and competitively destroy CTAB membrane forming Ag+ @CTAB-AuNPs complexes to result in enhanced peroxidase activity. Ag+ @CTAB-AuNPs show the highest peroxidase activity compared to similar-sized citrate-capped and ascorbic acid-capped AuNPs. Ag+ @CTAB-AuNPs can potentially develop into analyte-responsive systems and exhibit advantages in the optical sensing field. For example, the Ag+ @CTAB-AuNPs system shows an enhanced sensitivity and selectivity for acetylcholinesterase activity sensing compared to other methods.
RESUMEN
A fast (1 min), straightforward but efficient, click chemistry-based system that enables the rapid detection of free copper (Cu) ions in either biological fluids or living cells without tedious pretreatment is provided. Cu can quickly induce the conjugation between graphene oxide (GO) and a fluorescent dye via click reaction. On the basis of the high specificity of bioorthogonal reaction and the effective quenching ability of GO, the assay studied in this paper can respond to Cu ions in less than 1 min with excellent selectivity and sensitivity, which is the fastest sensor for Cu as far as it is known. In addition, the application of this system is verified by performing assays in living cells and untreated urine samples from patients suffering from Wilson's Disease. Such a Cu detection system shows great promises in both fundamental research and routine clinical diagnostics.
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Química Clic/métodos , Cobre/química , Grafito/químicaRESUMEN
Multiplexed analysis of molecules with different concentrations requires assays with a tunable detection range. A strategy is outlined that uses click chemistry to assemble horseradish peroxidase in a controlled fashion to generate enzyme assemblies as probes for multiplexed bioassays. This controllable assembly of enzymes on detection antibodies allows for lab-on-a-chip immunoassays with a tunable detection range from pg mL-1 to µg mL-1 . Simultaneous, multiplexed bioassays of clinically relevant inflammatory biomarkers in serum are demonstrated in one lab-on-a-chip format, with a limit of detection of 0.47â pg mL-1 for interleukin-6, 2.6 pg mL-1 for procalcitonin, and 40â ng mL-1 for C-reactive protein. This controlled assembly technique provides a multiplexed platform for simultaneous and quantitative analyses of both low-abundance and high-abundance biomarkers with a broad detection range, which holds great promise as a point-of-care platform for biomedical diagnostics.
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Bioensayo/métodos , Enzimas/metabolismo , Dispositivos Laboratorio en un Chip , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Humanos , Inflamación/sangre , Interleucina-6/metabolismo , Límite de Detección , Sondas Moleculares , Polipéptido alfa Relacionado con Calcitonina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We report an ultrasensitive, quantitative, and rapid bioluminescent immunosensor (ABS) for point-of-care testing (POCT) of the disease biomarker in clinical samples using double enzymes including alkaline phosphatase (ALP) and luciferase. In the presence of the biomarker, the ALP attached on the surface of immuno-nanocomplex dephosphorylates adenine triphosphate (ATP), subsequently inhibiting the ATP-luciferin-luciferase bioluminescent reaction. The highly sensitive response of ATP (picomolar level) allows for ultrasensitive detection of biomarker via the effective change of the bioluminescence intensity through ALP- and luciferase-catalyzed reactions, which can be quantitatively determined by a portable ATP detector. This ABS fulfills the criteria for POCT that performs sensitive (femtomolar level of biomarkers) and quantitative measurement quickly (less than 1 h) with minimal equipment (portable detector).
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Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Adenosina Trifosfato/análisis , Fosfatasa Alcalina/química , Biocatálisis , Biomarcadores/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Límite de Detección , Luciferasas/química , Mediciones Luminiscentes/instrumentación , Nanopartículas del Metal/química , Pruebas en el Punto de Atención , Poliestirenos/química , Polipéptido alfa Relacionado con Calcitonina/análisisRESUMEN
Bacterial infections, especially multidrug-resistant bacterial infections, are an increasingly serious problem in the field of wound healing. Herein, bacterial cellulose (BC) decorated by 4,6-diamino-2-pyrimidinethiol (DAPT)-modified gold nanoparticles (Au-DAPT NPs) is presented as a dressing (BC-Au-DAPT nanocomposites) for treating bacterially infected wounds. BC-Au-DAPT nanocomposites have better efficacy (measured in terms of reduced minimum inhibition concentration) than most of the antibiotics (cefazolin/sulfamethoxazole) against Gram-negative bacteria, while maintaining excellent physicochemical properties including water uptake capability, mechanical strain, and biocompatibility. On Escherichia coli- or Pseudomonas aeruginosa-infected full-thickness skin wounds on rats, the BC-Au-DAPT nanocomposites inhibit bacterial growth and promote wound repair. Thus, the BC-Au-DAPT nanocomposite system is a promising platform for treating superbug-infected wounds.
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Celulosa/química , Bacterias Gramnegativas/efectos de los fármacos , Nanopartículas del Metal/química , Enfermedades de la Piel/microbiología , Cicatrización de Heridas/efectos de los fármacos , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Nanocompuestos/química , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Ratas , Enfermedades de la Piel/tratamiento farmacológicoRESUMEN
We developed a competitive colorimetric nanosensor based on Au@Ag bimetallic nanoparticles for the detection of interleukin-6 (IL-6) in clinical samples. Antibody-conjugated magnetic nanoparticles (MNPs) and polystyrene (PS) microparticles conjugated with both catalase and a secondary antibody can form sandwich structures that enable one-step target enrichment and separation. The catalase on the surface of the PS can catalyze the hydrolysis of hydrogen peroxide (H2O2) to regulate the deposition of Ag+ on the surface of gold nanoparticles (AuNPs), and forms different sizes and amounts of Au@Ag bimetallic nanoparticles (Au@AgNPs) which produce a distinct color signal for readout with the naked eye. Our sensor features high sensitivity, selectivity, reproducibility and anti-interference property as a result of comprehensive parameter optimization. The limit of detection of IL-6 can reach 11 pg mL-1 with the naked eye and 1.2 pg mL-1 by quantitative instrumental analysis. The whole analysis can be finished within 1 h. More importantly, we successfully apply our platform or the detection of IL-6 in clinical samples with better accuracy than conventional enzyme-linked immunosorbent assay (ELISA).
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Biomarcadores/sangre , Colorimetría , Interleucina-6/sangre , Nanopartículas del Metal , Oro , Humanos , Peróxido de Hidrógeno , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , PlataRESUMEN
This report describes a colorimetric assay for trivalent metal cations (M(3+)) using gold nanoparticles (AuNPs)-modified with oppositely charged thiols that can form intermolecular zwitterionic surfaces. Zwitterionic AuNPs (Zw-AuNPs) are stable in high-salt solutions and well-dispersed in a wide range of pH values. M(3+) including Fe(3+), Al(3+), and Cr(3+) can effectively trigger the aggregation of Zw-AuNPs by interfering with their surface potential, and aggregated AuNPs can be regenerated and recycled by removing M(3+). In our approach, the output signal can be observed by the naked eye within a micromolar (µM) concentration range. Uniquely, our assay is capable of discriminating Fe(3+) from Fe(2+), which is challenging using traditional approaches. More importantly, Zw-AuNPs can be stored stably at room temperature for a long period (3 months) with constant detection performance. Both the cost-effectiveness and the long shelf life make Zw-AuNPs ideal for detecting M(3+) in resource-poor and remote areas.
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The boom of nanotechnology has yielded exciting developments in designing new kinds of colorimetric immunoassays. These nanomaterial-associated immunoassays have shown great potential for clinical translation and a number of them have already been implemented for testing patient samples from the clinics. Different from most reviews where researchers typically focus on a specific type of nanomaterial or describe assays based on the types of materials, we classify these assays by the function of nanomaterials, focusing on reviewing the distinct phenomenon of nanomaterials and how these properties are utilized to overcome limitations faced by traditional colorimetric immunoassays. We also discuss the challenges and give our perspectives in this field.
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Colorimetría/métodos , Inmunoensayo/métodos , Nanoestructuras , Nanotecnología/métodos , Animales , Humanos , Nanoestructuras/químicaRESUMEN
We report a method for the rapid and efficient identification of bacteria making use of five probes having fluorescent characteristics (F-array) and subsequent statistical analysis. Eight kinds of bacteria, including normal and multidrug-resistant bacteria, are differentiated successfully. Our easy-to-perform and time-saving method consists of mixing bacteria and probes, recording fluorescent intensity data by automated flow cytometry, and statistical analysis. No washing steps are required in order to identify the different bacteria simultaneously.