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Phosphate plays a crucial role in microbial proliferation, and the regulation of the phosphate concentration can modulate the fermentation efficiency. In this study, based on Lambert-Beer's Law and the selective absorption characteristics of substances under light, a dual-light-type photoelectric colorimetric device for phosphate determination was designed. The device's main components, such as the excitation light path and incubation stations, were modeled and simulated. The primary performance of the instrument was verified, and comparative experiments with a UV-1780 spectrophotometer were conducted to validate its performance. The experimental results demonstrate that this device exhibits a high degree of linearity with an R2 value of 0.9956 and a repeatability of ≤1.72%. The average temperature rise rate at the incubation stations was measured at 0.44 °C/s, with a temperature uniformity ≤ ±0.1 °C (temperature set at 37.3 °C). Consistently observed trends in the measurement of 23 CHO cell suspensions using the UV-1780 spectrophotometer further validated the accuracy and reliability of the device's detection results.

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Enzyme-based biosensors commonly utilize the drop-casting method for their surface modification. However, the drawbacks of this technique, such as low reproducibility, coffee ring effects, and challenges in mass production, hinder its application. To overcome these limitations, we propose a novel surface functionalization strategy of enzyme crosslinking via inkjet printing for reagentless enzyme-based biosensors. This method includes printing three functional layers onto a screen-printed electrode: the enzyme layer, crosslinking layer, and protective layer. Nanomaterials and substrates are preloaded together during our inkjet printing. Inkjet-printed electrodes feature a uniform enzyme deposition, ensuring high reproducibility and superior electrochemical performance compared to traditional drop-casted ones. The resultant biosensors display high sensitivity, as well as a broad linear response in the physiological range of the serum phosphate. This enzyme crosslinking method has the potential to extend into various enzyme-based biosensors through altering functional layer components.

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Excessive phosphate poses a serious ecological and human health risk, and thereby, monitoring its trace concentration is of great significance to environmental protection and human health. In this work, a zirconium-porphyrin framework (PCN-222) with excellent stability and unique luminescence properties was designed to modify the surface of the indium tin oxide electrode, which was first used as a photoelectrochemical (PEC) probe for phosphate detection. The PCN-222-modified PEC probe demonstrated an excellent selectivity and stability and indicated a linear response to phosphate in the range of 0-106 nM with a limit of detection (LOD) as low as 1.964 nM. To the best of our knowledge, this is the phosphate probe with the lowest LOD, and this is also the first signal-on PEC probe toward phosphate based on PCN-222. More importantly, the PEC probe can be validated for the good applicability of trace phosphate detection in real water samples, indicating a good application prospect. Finally, a series of electrochemical and spectroscopic studies have proved that phosphate can bind to the indium tin oxide (ITO)/PCN-222 electrode, which shortens the distance of the space charge region while reducing the bandwidth and thus facilitates the transfer of photogenerated electrons across the energy band barrier to reduce O2 in the electrolyte, producing an enhanced cathodic photocurrent signal. The proposed strategy of the highly sensitive PEC probe provides a promising platform for more effective label-free phosphate monitoring in the environment and organisms.

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Triphenyl phosphate (TPHP), a typical kind of organophosphorus flame retardants (OPFRs) with aryl groups, has been recognized as an emerging contaminant that causes environmental and health hazards. It is a pervasive threat that can be frequently detected in the environment and living organisms. Hence, establishing an efficient analytical method for TPHP is an urgent issue. In this work, a heteropolyacid (HPA)-luminol chemiluminescence strategy coupled with UV-assisted persulfate (PS) activation was proposed for the sensitive and selective detection of TPHP. The UV-assisted PS oxidation pretreatment could decompose the water-insoluble TPHP into smaller orthophosphates, which were further converted into HPA with the subsequently introduced vanadium­molybdenum acid. The formed HPA served as a catalyst to oxidize luminol, and strong chemiluminescence at 425 nm was generated immediately. Furthermore, the degradation process of TPHP and chemiluminescence mechanism were also investigated. The results demonstrated that some reactive oxygen radicals such as SO4-, OH, 1O2, and O2-, were involved in the degradation and chemiluminescence reaction. Notably, this proposed chemiluminescence analytical strategy realized a highly sensitive detection for TPHP, and granted the limit of detection down to 0.38 ppt. This study provides an attractive perspective for the detection of emerging OPFRs.

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Concerns for agri-food safety and environmental management require development of simple to use and cost- and time-effective multiplex sensors for point-of-need (PON) chemical analytics by public end-user. Simultaneous detection of nitrates, phosphates, and pH is of importance in soil and water analysis, agriculture, and food quality assessment. This article demonstrates a suite of stainless steel microneedle electrochemical sensors for multiplexed measurement of pH, nitrate, and phosphate using faradaic capacitance derived from cyclic voltammetry as the mode of detection. The multi-target microneedle sensors were fabricated by layer-by-layer (LbL) assembly in a stainless steel hypodermic microneedle substrate. For nitrate sensing, the stainless steel was coated with carbon nanotube/cellulose nanocrystal (CNT)/CNC) decorated with silver nanoparticles (Ag). For pH measurement, the polyaniline (pANI) was coated onto the CNT/CNC@Ag film, while for phosphate detection, the CNT/CNC/Ag @pANI microneedle was further decorated with ammonium molybdenum tetrahydrate (AMT). The microelectrode platforms were characterized by FTIR, Raman, and microscopic techniques. The nitrate- and phosphate-based microneedle electrochemical sensors had excellent selectivity and sensitivity, with a determined limit of detection (LOD) of 0.008 mM and 0.007 mM, respectively. The pH microneedle sensor was responsive to pH in the linear range of 3-10. The three microneedle sensors yielded repeatable results, with a precision ranging from 4.0 to 7.5% RSD over the concentration ranges tested. The inexpensive (~ 1 $ CAD) microneedle sensors were successfully verified for use in quantification of nitrate, pH, and phosphate in brewed black coffee as a real sample. As such, the microneedle sensors are economical devices and show great promise as robust platforms for PON precision chemical analytics.

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Nutrient pollution remains one of the greatest threats to water quality and imposes numerous public health and ecological concerns. Phosphate, the most common form of phosphorus, is one of the key nutrients necessary for plant growth. However, phosphate concentration in water should be carefully monitored for environmental protection requirements. Hence, an easy-to-use, field-deployable, and reliable device is needed to measure phosphate concentrations in the field. In this study, an inexpensive dip strip is developed for the detection of low concentrations of phosphate in water and seawater. In this device, ascorbic acid/antimony reagent was dried on blotting paper, which served as the detection zone, and was followed by a wet chemistry protocol using the molybdenum method. Ammonium molybdate and sulfuric acid were separately stored in liquid form to significantly improve the lifetime of the device and enhance the reproducibility of its performance. The device was tested with deionized water and Sargasso Sea seawater. The limits of detection and quantification for the optimized device using a desktop scanner were 0.134 ppm and 0.472 ppm for phosphate in water and 0.438 ppm and 1.961 ppm in seawater, respectively. The use of the portable infrared lightbox previously developed at our lab improved the limits of detection and quantification by a factor of three and were 0.156 ppm and 0.769 ppm for the Sargasso Sea seawater. The device's shelf life, storage conditions, and limit of detection are superior to what was previously reported for the paper-based phosphate detection devices.

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Rapid and accurate detection of phosphate (Pi) in complex biological fluid is of critical importance for timely warning of Pi accumulation and monitoring Pi related pathological process. Up to date, various luminescent probes have been developed for Pi determination in aqueous media. However, the huge obstacles of the current probes suffer from the inherent issues such as time-consuming, tedious preparation and unavoidable background interference during Pi detection. To circumvent this limitation, we proposed a universal and facile strategy to fabricate a novel sensitizer-Ln3+@surfactant micelle probe with time-resolved luminescent (TRL) superiority through the self-assembly of sensitizer, Ln3+ and surfactant. Through this design, the sensitizer-Ln3+ chelate can be encapsulated into the surfactant constructed micelle and Ln3+ luminescence can be substantially lighted up through the effective energy transfer from the coordinated sensitizer and the assistance of Triton X-100. Such high TRL signal can be sensitively and specifically quenched by Pi, which was attributed to the specific coordination competition between sensitizer and Pi towards Ln3+. Benefitting from the background-free interference and highly sensitive TRL response of the sensitizer-Ln3+@surfactant probe, we achieved the rapid, selective and sensitive detection of Pi in the range of 0.5-120 µM with a limit of detection (LOD) of 0.19 µM. Furthermore, the accuracy of the proposed method based on the Ln3+ involved micelle probes was further verified through the quantitation of Pi in real biological samples.

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Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a single turnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.

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Phosphate (Pi) not only plays a significant role in physiological processes, but also is an important indicator for aquatic ecosystems. The dual-functional lanthanide metal organic frameworks (MOFs) were synthesized for visual and ultrasensitive ratiometric fluorescent detection of Pi based on aggregation-induced energy transfer. In the MOFs material, ciprofloxacin (CIP) functions as an energy donor and results in the fluorescence enhancement of Eu3+; the introduction of pyromellitic acid can cause the aggregation of the CIP-Eu3+ complex, and red characteristic fluorescence of Eu3+ at 614 nm is further enhanced (about 40 times). When Pi is added to the MOFs solution, CIP is released from the MOFs, red fluorescence of Eu3+ is quenched and blue fluorescence of CIP is simultaneously recovered, thereby a ratiometric fluorescent probe for the detection of Pi was fabricated. The fluorescent response based on intermolecular energy transfer of the CIP-Eu3+ complex is very sensitive to Pi. The limit of detection (3σ/K) of the probe is ultrasensitive and attains 4.4 nM. The possible interferential substances such as 17 common metal ions and 14 anions investigated do not interfere with the Pi detection. The ratiometric fluorescent probe has been successfully used in the determination of Pi in real human urine and lake water samples. This work may supply a new strategy for fabricating ratiometric fluorescent probe and a prospective application in biological and environmental samples.

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High concentrations of certain nutrients, including phosphate, are known to lead to undesired algal growth and low dissolved oxygen levels, creating deadly conditions for organisms in marine ecosystems. The rapid and robust detection of these nutrients using a colorimetric, paper-based system that can be applied on-site is of high interest to individuals monitoring marine environments and others affected by marine ecosystem health. Several techniques for detecting phosphate have been reported previously, yet these techniques often suffer from high detection limits, reagent instability, and the need of the user to handle toxic reagents. In order to develop improved phosphate detection methods, the commonly used molybdenum blue reagents were incorporated into a paper-based, colorimetric detection system. This system benefited from improved stabilization of the molybdenum blue reagent as well as minimal user contact with toxic reagents. The colorimetric readout from the paper-based devices was analyzed and quantified using RGB analyses (via ImageJ), and resulted in the detection of phosphate at detection limits between 1.3 and 2.8 ppm in various aqueous media, including real seawater.

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Here, a portable and accurate phosphate sensor using a gradient Fabry-Pérot array (FPA) is proposed. It can form a bidirectional gradient concentration (absorbance) distribution in the gradient FPA, simplifying the complex operations to get a standard curve and saving time. The gradient FPA can effectively filter out the interference (bubbles, light intensity, and salinity) while improving the absorbance, achieving a highly accurate and stable detection. Besides, the smartphone simplifies data processing and makes sensors more portable. In this work, the detection errors of standard solutions (100, 50, and 30 µM) are 0.39, 1.48, and 1.84%, respectively, and it has also been demonstrated with errors of 2.46 (sample 1, seawater), 2.08 (sample 2, lake water), and 1.83% (sample 3, sewage) for natural samples detection, which is more accurate than a traditional analyzer. The sensor has a good performance when affected by bubbles, light intensity, and salinity. In addition, the detection time is shortened to 80 s, which is more time saving compared with traditional devices, and the limit of detection (LOD) is 0.4 µM. It can be predicted that the novel optofluidic sensor is conducive to build a smart nutrient monitoring system and will be applied in the field of biochemistry and environmental chemistry.

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Porphin-based carbon dots (denoted as PCDs) are prepared through a one-step hydrothermal method by using meso-tetra (4-carboxyphenyl) porphin (TCPP), citric acid, and ethanediamine as precursor. PCDs give rise to the optimal photoluminescence at λex/λem = 375/645 nm, exhibit an excitation-independent property, excellent water solubility, and good biocompatibility, which provide red emission and avoid the autofluorescence as an efficient fluorescent imaging probe. On the other hand, when Eu3+ is added into PCDs, the carboxylate groups located on the surface of PCDs exhibit high affinity to Eu3+, resulting in the fluorescence of PCDs turning off via static quenching. In the presence of phosphate, owing to the strong coordination with Eu3+, the fluorescence of PCDs turns on. Based on this performance, a novel "turn off-on" phosphate sensing system is developed. The detection limit of this sensing system can attain 3.59 × 10-3 µmol L-1. This system has been utilized for the detection of phosphate in real samples successfully, which further demonstrates potential applications in biological diagnostic and environmental analysis.

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A composite probe has been developed for fluorometric determination and imaging of phosphate in real water samples and in cells. The method is based on the use of weakly blue fluorescent bromine-doped carbon dots (C-dots) containing aromatic carbon-bromine groups and loaded with Fe3+ ions. The carboxy, phenolic hydroxy and aldehyde groups on the surface of the C-dots can coordinate with Fe3+ to form an adsorbed complex that reduces the blue fluorescence through an inner filter effect. If phosphate is added, it will capture Fe3+ on the surface of C-dots and restore fluorescence by ~88% via a displacement approach. The probe, best operated at excitation/emission maxima of 370/418 nm, has a linear response in the 0.4 to 22 µM phosphate concentration range and a 0.25 µM of detection limit. The relative standard deviation (at a phosphate level of 8.0 µM) is 3.6% (for n = 5). The method was applied to confocal imaging of phosphate in HeLa cells. Graphical abstractSchematic representation of the synthesis of bromine-doped carbon dots (C-dots) by a "one-step" approach. They are shown to be capable of (a) detecting phosphate in real water samples through the displacement approach, and (b) of imaging intracellular phosphate.

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Carbon quantum dots (CQDs), prepared by one-step hydrothermal treatment of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) and triethylamine (TEA), could be exfoliated or delaminated into single-layered graphene quantum dots (s-GQDs) with methanol for the first time, with fluorescence (FL) emission at 500 nm when excited at 417 nm. The s-GQDs, with more sufficient carboxyl groups on the surface than CQDs, could be induced to be aggregated by metal ion dysprosium (Dy3+), resulting in aggregation-induced emission quenching effect subsequently. However, the presence of phosphate (PO43-) destroys the Dy3+-induced aggregates of s-GQDs owing to the strong coordination between Dy3+ and PO43-, inducing the FL emission recovery of the s-GQDs and providing selective detection method of PO43- in the artificial wetlands with the linear range of 0.2-30 µM and determination limit of 0.1 µM (3σ).

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This work attempted to prolong the validity of the molybdate mixed solution and ascorbic acid solution used in the phosphomolybdenum blue spectrophotometric method by improving their preservation according to the influence factors. The results showed that the molybdate mixed solution can be directly preserved in darkness with validity over half a year. The ascorbic acid solution is influenced by light, temperature, pH, metal ions, oxygen, and bacteria. The validity of ascorbic acid is shortened as the temperature rises. Through keeping in darkness, adding complexing agents, adjusting pH, removing oxygen and sterilization, the validity of ascorbic acid solution was prolonged to over 2.7 times under 4 °C and over 5 times under 25 °C. At the same time, the hybrid solution of ascorbic acid solution and molybdate mixed solution should be preserved separately, otherwise the using effect is poor.

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Phosphate is not only an important indicator for aquatic ecosystems, but also plays vital roles in biosystems. A new strategy for ultrasensitive and selective detection of phosphate is fabricated based on a new insight found in this paper, in which a lower concentration of surfactant sodium dodecylbenzenesulfonate (SDBS) can greatly induce fluorescence resonance energy transfer (FRET) from ciprofloxacin (CIP) to Eu3+ in the CIP-Eu3+ complex. Surfactant SDBS does not act as a sensitizer for enhancing the fluorescence intensity of the system, but acts as a sensitizer of FRET and makes the native fluorescence of CIP quenched completely (switch off). Eu3+ ions can coordinate with the oxygen-donor atoms of phosphate, which weakens FRET from CIP to Eu3+ and results in the fluorescence recovery of CIP (turn on). The multicomplex of the CIP-Eu3+-phosphate has more sensitive fluorescent response than that of the reported coordination nanoparticle-based fluorescent probes. The LOD (S/N = 3) of this sensing system can attain 4.3 nM. The possible interferential substances existing in environmental samples, such as 17 common metal ions, 11 anions, and fulvic acid investigated, do not interfere with the phosphate detection. This sensing system has been successfully applied for phosphate detection in environmental samples such as wastewater, surface water, and atmospheric particulates. This work not only develops a fluorescent probe for the phosphate detection, but also provides a new strategy for designing fluorescent probes based on FRET or coordination nanoparticles.

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A novel fluorescence method for sensitive and selective detection of phosphate was developed based on near infrared emission Ag2S QDs/ Metal - Organic Shell Composite via the deposition of metal-organic (zinc-nitrogen) coordination shell around Ag2S QDs . Under optimal conditions described, the fluorescence intensity of the composite was decreased at 685 nm in the presence of phosphate, which was linearly related to the concentration of phosphate in the range of 0. 7 to 4.2 µM and 11.2 to 88.2 µM with the relative correlation coefficient of R2 = 0.998 and 0.987 respectively and detection limit as low as 6 nM. In addition, the proposed method was successfully utilized in serum samples, tap water and Yangtze River water samples with the recoveries ranged from 94.76 to 100.86 %, which presaged more opportunities for application in related bioassay and water sample researches.

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Fluorescence bioimaging potential, both in vitro and in vivo, of a yellow emissive triazole-based molecular marker has been investigated and demonstrated. Three different kinds of cells, viz Bacillus thuringiensis, Candida albicans, and Techoma stans pollen grains were used to investigate the intracellular zinc imaging potential of 1 (in vitro studies). Fluorescence imaging of translocation of zinc through the stem of small herb, Peperomia pellucida, having transparent stem proved in vivo bioimaging capability of 1. This approach will enable in screening cell permeability and biostability of a newly developed probe. Similarly, the current method for detection and localization of zinc in Gram seed sprouts could be an easy and potential alternative of the existing analytical methods to investigate the efficiency of various strategies applied for increasing zinc-content in cereal crops. The probe-zinc ensemble has efficiently been applied for detecting phosphate-based biomolecules.

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