Multi-wavelength deep-ultraviolet absorbance detector based upon program-controlled pulsing light-emitting diodes.

A novel approach for multi-wavelength ultraviolet (UV) absorbance detection has been introduced employing a single board computer (SBC) with a field programmable gate array (FPGA), Red Pitaya SBC, to generate separated micro pulses for three deep-ultraviolet light-emitting diodes (DUV-LEDs), λmax = 235, 250, and 280 nm, along with data acquisition and processing via a custom-made program. The pulse set generation and data acquisition were synchronized using the SBC. The outputs of the three pulsing DUV-LEDs were combined and transmitted to the flow cell via a solarisation resistant trifurcated optical fiber (OF). An ultra-fast responding photodiode was connected to the optical-fiber-compatible flow cell to record the intensity of the DUV pulses. Upper limit of detector linearity (A95 %) was found to be 1917 mAU, 2189 mAU, and 1768 mAU at 235 nm, 250 nm, and 280 nm, respectively, with stray light ≤0.9 %. In addition, the effective path length (Leff) was estimated to be ≥98.0 % of the length of the used flow cell (50 mm). The new pulsed multi-LEDs absorbance detector (PMLAD) has been successfully coupled with a standard liquid chromatograph and utilized for the analysis of pharmaceuticals. Paracetamol, caffeine, and aspirin were simultaneously determined at 250, 280, and 235 nm, respectively, using the PMLAD. The absorbance ratios between the different wavelengths were applied to further confirm the identity of the studied compounds. Excellent linearity was achieved over a range of 0.1-3.2 µg/mL for paracetamol, 0.4-6.4 µg/mL for caffeine, and 0.8-12.8 µg/mL for aspirin with a regression correlation coefficient (r2) ≥ 0.99996. The quantitation limits (LOQs) were 0.10 µg/mL, 0.38 µg/mL, and 0.66 µg/mL for paracetamol, caffeine, and aspirin, respectively.

Recent advances in miniaturization of portable liquid chromatography with emphasis on detection.

Liquid chromatography is a prominent analytical technique in separation science and chemical analysis, applied across numerous fields of research and within industrial applications. Over the past few decades, there has been a growing interest in the miniaturization of this technique, which has been particularly enabled through new miniature and portable detection technologies for in-field, at-site, and point-of-need (collectively 'out-of-lab') analyses. Accordingly, significant advances have been made in recent years in the development of miniaturized liquid chromatography with photometric, electrochemical, and mass spectrometric detection, enabling the development of field-deployable and portable instruments for various applications. Herein, recent developments in the miniaturization of detection systems for inclusion within, and/or coupling with, portable liquid chromatographic systems, are reviewed in detail together with critical comments and expected future trends in this area.

Rapid and selective screening of organic peroxide explosives using acid-hydrolysis induced chemiluminescence.

Organic peroxide explosives (OPEs) are unstable, non-military, contemporary security threats often found in improvised explosive devices. Chemiluminescence (CL) can be used to detect OPEs, via radical formation consisting of peroxide moieties (-O-O-) under acidic conditions. However, selectivity for specific OPEs is hampered by the ubiquitous background of H2O2. Herein, we report the differentiation of hexamethylene triperoxide diamine (HMTD), triacetone triperoxide (TATP), and methyl ethyl ketone peroxide (MEKP) by specific flow injection analysis-CL (FIA-CL) signal profiles, after H2SO4 treatment. The radical degradation pathway of each structure, and its corresponding FIA-CL profile, was explored using mass spectrometry to reveal the rapid loss of -O-O- from TATP and HMTD structures, while MEKP formed CL signal-sustaining oligomers, as opposed to the immediate attenuation of H2O2. The CL response for OPEs in an aqueous media, measured via the described FIA-CL method, enabled ultra-trace limits of detection down to 0.40 µM for MEKP, 0.43 µM for HMTD, and 0.40 µM for TATP (combined linear range 1-83 µM with 95% confidence limit, n = 12). Expanded uncertainties of measurement (UM) of MEKP = ±0.98, HMTD = ±1.03, and TATP = ±1.1 (UM included probabilities of false positive and false negative as well as standard deviations of % recoveries and limit of detections of OPEs). Direct aqueous sample introduction via FIA-CL thus offers the prospect of rapid and selective screening of OPEs in security-heightened settings (e.g., airports), averting false positives from more ubiquitous H2O2.

Microfluidic paper-based fluorescence sensor for L-homocysteine using a molecularly imprinted polymer and in situ-formed fluorescent quantum dots.

Microfluidic paper-based analytical devices modified with molecularly imprinted polymers (µPADs@MIPs) were developed for fluorescent detection of targeted thiols via in situ UV-induced formation of quantum dots (µPADs@MIPs@QDs). The selectivity enhancement by the MIP layer formed on the filter paper surface was demonstrated for the isolation of L-homocysteine from wine. Followed by the addition of metal precursors solution (Zn/Cd/Cu) and UV irradiation, fluorescent quantum dots were formed thus enabling quantitative detection of the thiol (serving as a QD capping agent). The effect of different semiconductors was investigated to achieve a lower band gap and higher fluorescence intensity. Increasing fluorescence intensity in the presence of thiol groups was obtained for the following precursors mixture composition: ZnCdCu/S > ZnCd/S > ZnCu/S > ZnS. The proposed method has a good relationship between the fluorescence intensity of ZnCdCu/S QDs and L-homocysteine in a linear range from 0.74 to 7.40 µM with a limit of detection (LOD) and quantification (LOQ) of 0.51 and 1.71 µM respectively. This method was applied for the determination of L-homocysteine in white wine with RSD under 6.37%.

Detection of pesticides in food products using paper-based devices by UV-induced fluorescence spectroscopy combined with molecularly imprinted polymers.

In this proof-of-concept study, we explore the detection of pesticides in food using a combined power of sensitive UV-induced fingerprint spectroscopy with selective capture by molecularly imprinted polymers (MIPs) and portable cost-effective paper-based analytical devices (PADs). The specific pesticides used herein as model compounds (both pure substances and their application products for spraying), were: strobilurins (i.e. trifloxystrobin), urea pesticides (rimsulfuron), pyrethroids (cypermethrine) and aryloxyphenoxyproponic acid herbicides (Haloxyfop-methyl). Commercially available spraying formulations containing the selected pesticides were positively identified by MIP-PADs swabs of sprayed apple and tomato. The key properties of MIP layer - imprinting factor (IF) and selectivity factor (α) were characterized using trifloxystrobin (IF-3.5, α-4.4) was demonstrated as a potential option for in-field application. The presented method may provide effective help with in-field testing of food and reveal problems such as false product labelling.

Isotachophoresis for rapid transformation of Escherichia coli.

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2-3 × 105 ) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 µm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (-16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.

UV-induced Zn:Cd/S quantum dots in-situ formed in the presence of thiols for sensitive and selective fluorescence detection of thiols.

In this work, we explored a new approach to a simple and sensitive fluorescence detection of thiols. The approach takes advantage of an in-situ formation of UV light-induced fluorescent nanoparticles (ZnCd/S quantum dots), while utilizing the thiol group of the analyte as a capping agent. The selectivity is ensured by the selective isolation of the thiol analyte by a polydopamine molecularly imprinted polymeric (MIP) layer. Based on this approach, a method for determination of thiols was designed. Key experimental parameters were optimized, including those of molecular imprinting and of effective model thiol molecule (L-cysteine) isolation. The relationship between the fluorescence intensity of ZnCd/S quantum dots and the concentration of L-cysteine in the range of 12-150 µg/mL was linear with a detection limit of 3.6 µg/mL. The molecularly imprinted polymer showed high absorption mass capacity (1.73 mg/g) and an excellent selectivity factor for L-cysteine compared to N-acetyl-L-cysteine and L-homocysteine of 63.56 and 87.48, respectively. The proposed method was applied for L-cysteine determination in human urine with satisfactory results. Due to a high variability of molecular imprinting technology and versatility of in-situ probe formation, methods based on this approach can be easily adopted for analysis of any thiol of interest.

Metallothionein dimerization evidenced by QD-based Förster resonance energy transfer and capillary electrophoresis.

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd2+ ions between molecules of metallothionein, the QDs and Cy3 were close enough, enabling a FRET signal. The FRET efficiency was calculated to be in the range of 11-77%. The formation of MT dimers in the presence of Cd2+ ions was confirmed by MALDI-MS analyses. Finally, the process of oligomerization resulting in FRET was monitored by CE, and oligomerization of MT was confirmed.

Miniature Multiwavelength Deep UV-LED-Based Absorption Detection System for Capillary LC.

A new miniature deep UV absorbance detector has been developed using low-cost and high-performance LEDs, which can be operated in both scanning (230 to 300 nm) and individual wavelength (240, 255, and 275 nm) detection modes. The detector is mostly composed of off-the-shelf components, such as LEDs, trifurcated fiber optic assembly, a capillary Z-type flow cell, and photodiodes. It has been characterized for use with a standard capillary LC system and was benchmarked against a standard variable wavelength capillary LC detector. The detector shows very low levels of stray light (<0.4%), utilization of up to 99.0% of the effective path length of the flow cell, a wide dynamic range (0.5 to 200 µg/mL for sulfamethazine, carbamazepine, and flavone), and low noise levels (at 300 µAU level). The detector was applied within a miniaturized LC system.

Miniaturized LC in Molecular Omics.

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Continuous and real-time indoor and outdoor methane sensing with portable optical sensor using rapidly pulsed IR LEDs.

We designed a simple, portable, low-cost and low-weight nondispersive infrared (NDIR) spectroscopy-based system for continuous remote sensing of atmospheric methane (CH4) with rapidly pulsed near-infrared light emitting diodes (NIR LED) at 1.65 µm. The use of a microcontroller with a field programmable gate array (µC-FPGA) enables on-the-fly and wireless streaming and processing of large data streams (~2 Gbit/s). The investigated NIR LED detection system offers favourable limits of detection (LOD) of 300 ppm (±5%) CH4,. All the generated raw data were processed automatically on-the-fly in the µC-FPGA and transferred wirelessly via a network connection. The sensing device was deployed for the portable sensing of atmospheric CH4 at a local landfill, resulting in quantified concentrations within the sampling area (ca 400 m2) in the range of 0.5%-3.35% CH4. This NIR LED-based sensor system offers a simple low-cost solution for continuous real-time, quantitative, and direct measurement of CH4 concentrations in indoor and outdoor environments, yet with the flexibility provided by the custom programmable software. It possesses future potential for remote monitoring of gases directly from mobile platforms such as smartphones and unmanned aerial vehicles (UAV).

Radiometric characterisation of light sources used in analytical chemistry - A review.

Light sources are an indispensable component of an overwhelmingly large number of analytical methods. Radiometric characterisation of light sources in analytical chemistry is therefore of fundamental importance. This review presents up to date knowledge on methods to characterise radiometric properties of light sources in terms of radiometric power, irradiance, brightness, luminous efficacy, luminous efficiency and emission spectra, all of which are crucial parameters for their use in analytical chemistry. Special attention is paid to radiometric characterisation of new generations of light sources with focus on miniaturised and low-cost light sources suitable for portable analytical instrumentation. Miniaturised light sources, especially new generations of solid-state light sources including solution processable quantum dot light emitting diodes (QLEDs), organic LEDs (OLEDs) as well as conventional LEDs and lasers, are radiometrically characterised through various spectrophotometric, actinometric as well as new facile radiometric methods. Although the areas of analytical use of new light sources including QLEDs, OLEDs as well as other important light sources such as deep ultraviolet (DUV) and infrared LEDs in analytical chemistry are yet to reach their potential, their radiometric characterisation opens future options for their wider deployment in analytical chemistry.

Distance-based paper device using polydiacetylene liposome as a chromogenic substance for rapid and in-field analysis of quaternary ammonium compounds.

This work presents an affordable distance-based microfluidic paper-based device (µPAD), using polydiacetylene (PDA) liposome as a chromogenic substance with a smartphone-based photo editor, for rapid and in-field analysis of quaternary ammonium compounds (QACs) (e.g., didecyldimethylammonium chloride (DDAC), benzyldimethyltetradecyl ammonium chloride (BAC), and cetylpyridinium chloride (CPC)). In-field analysis of these compounds is important to ensure their antimicrobial activity and user safety since they are widely utilized as disinfectants in households and hospitals. The µPAD featured a thermometer-like shape consisting of a sample reservoir and a microchannel as the detection zone, which was pre-deposited with PDA liposome. The color change from blue to red appeared in the presence of QACs and the color bar lengths were proportional to the QAC concentrations. Reactions of QACs with the PDA required a specific pH range (from pH 4.0 to 10.0) and a readout time of 7 min. Analytical performance characteristics of the device were tested with DDAC, BAC, and CPC showing acceptable specificity, accuracy (96.1-109.4%), and precision (%RSDs ≤ 9.3%). Limits of detection and quantitation were in the ranges of 20 to 80 and 70 to 250 µM, respectively. Feasibility of the newly developed device was demonstrated for in-field analysis of QACs in fumigation solution providing comparable results with those obtained from a colorimetric assay (P > 0.05). The proposed device shows potentials for further applications of other analytes since it offers speed, simplicity, and affordability for in-field analysis, especially in remote areas where expertise, resources, and infrastructures are limited. Graphical abstract.

Miniature and fully portable gradient capillary liquid chromatograph.

A robust, portable and miniature battery powered gradient capillary liquid chromatograph (total weight ∼2.7 kg, without battery ∼2.0 kg), with integrated microfluidic injection, column heating and high sensitivity low-UV absorbance detection is presented. The portable capillary chromatograph, was applied with a packed reversed-phase capillary column (100 mm × 300 µm I.D., 5 µm ODS), housed within an integrated capillary column heater controlled by a proportional-integral-derivative (PID) chip module. The system delivered retention time and peak area relative standard deviation in isocratic mode of <0.7% (n = 10) and <3.3% (n = 10), respectively, and <0.1% (n = 10) and <2.3% (n = 10) respectively, for gradient elution mode. Detection was based upon a 255 nm light-emitting diode (LED) using one of two commercial capillary flow-cell options, namely a high sensitivity 12 nL Agilent capillary z-cell (HSDC) and a 45 nL Thermo Fisher Scientific UZ-View™ flow cell (UZFC). The HSDC, housed within a 3D printed detector arrangement, gave an effective pathlength of 1.01 mm (84% of nominal pathlength) and stray light of only 0.2%. Limits of detection for four test small molecule pharmaceuticals ranged from 65 to 101 µg L-1 based upon a 316 nL injection volume, with separation efficiencies of between 18,000 and 29,700 N m-1, with sub-4 min run times. The portable capillary LC system was successfully coupled to a small footprint portable mass spectrometer (Microsaic 4500 MiD) to demonstrate compatibility and 'point-of-need' miniaturised LC-MS capability.

One step multi-material 3D printing for the fabrication of a photometric detector flow cell.

Optical detection is the most common detection mode for many analytical assays. Photometric detection systems and their integration with analytical systems usually require several assembly parts and manual alignment of the capillary/tubing which affects sensitivity and repeatability. 3D printing is an innovative technology for the fabrication of integrated complex detection systems. One step multi-material 3D printing has been explored to fabricate a photometric detector flow cell from optically transparent and opaque materials using a dual-head FDM 3D printer. Integration of the microchannel, the detection window and the slit in a single device eliminates the need for manual alignment of fluidic and optical components, and hence improves sensitivity and repeatability. 3D printing allowed for rapid design optimisation by varying the slit dimension and optical pathlength. The optimised design was evaluated by determining stray light, effective path length and the signal to noise ratio using orange G. The optimised flow cell with extended path length of 10 mm and 500 µm slit yielded 0.02% stray light, 89% effective path length and detection limit of 2 nM. The sensitivity was also improved by 80% in the process of optimisation, using a blue 470 nm LED as a light source.

Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement.

Cytochrome P450 (CYP450), and in particular CYP3A4, is the most abundantly expressed CYP450 isozyme implicated in many drug-drug and medicinal plant-drug interactions. Therefore, incorporation of CYP3A4 enzyme screening at an early stage of drug discovery is preferable in order to avoid enzymatic interactions. Here we present for the first time a paper-based CYP3A4 immobilized sol-gel-derived a platform using resorufin benzyl ether as a fluorogenic enzyme substrate used to investigate enzyme activity. The fluorescence intensity of the product can be simply quantified by using a handheld digital microscope and an image analysis software. The limit of quantitation was 0.35 µM with good precision (RSDs < 4.1%). Furthermore, the assay of CYP3A4 activity on the developed paper-based device provided comparable results with those obtained from conventional well-plates (p > 0.05), while offering simplicity and lower cost. Kinetic parameters of the immobilized CYP3A4 in sol-gel coated paper were calculated from the Lineweaver-Burk plot, including Michaelis constant (Km) and maximum velocity (Vmax), which were 2.71 ±â€¯0.35 µM and 0.43 ±â€¯0.05 µM/min, respectively. Moreover, a functional test of these devices was conducted by assessments of known CYP3A4 inhibitors (i.e. ketoconazole, itraconazole) and inducers (i.e. phenytoin, carbamazepine). To further demonstrate the broad range of uses, the devices were utilized to assay plant extracts i.e. Areca catechu seeds, Camellia sinensis leaves, Eclipta prostrata aerial part, providing results in good agreement with previous studies. Furthermore, the sol-gel immobilized enzyme stored at 4 °C can increase storage stability, offering the activity of 86.3 ±â€¯0.4% after 3-weeks storage, equivalent to the activity of the free enzyme solution after 1-week storage. The developed paper-based devices offer versatility, portability and low-cost.

Ion-Exchange Based Immobilization of Chromogenic Reagents on Microfluidic Paper Analytical Devices.

Distance-based detection methods, as used in development of microfluidic paper analytical devices (µPADs), rely on the dynamic formation of a colored band along the length of the paper microfluidic channels. The color change is driven by the reaction of chromogenic reagents (typically water-insoluble) that are bound to the paper, thus not subject to being washed away by the sample flow along the detection channel. Here, we introduce the use of an anion-exchange filter paper (as a replacement for standard, unmodified filter paper) for distance-based detection in µPADs, in order to immobilize the water-soluble anionic reagents upon the paper detection channels based on ion-exchange interactions of the oppositely charged paper (protonated tertiary amine groups) and the anionic groups of the reagents. The ion-exchange (IE) paper was initially characterized and its properties were compared with standard cellulose paper. The IE paper was shown to be capable of strong retention of anionic reagents exhibiting acidic functional groups (carboxylic, sulfonic), which become deprotonated and negatively charged when in contact with the IE paper. The effect of the ionic strength (10-250 mM Cl-) and pH (1-13) on the immobilization of the investigated reagents were also determined. The IE-µPADs were then modified with anionic chromogenic reagents and applied to distance-based determination of total calcium (LOD = 0.03 mM) and total acidity (LOD = 2.5 mM) content in serum and wine samples, respectively. The detailed mechanisms of the developed assays on the IE paper are also discussed. We propose that IE-µPADs represent a useful new addition to the distance-based detection toolbox and considerably enhance the applicability of such a detection method.

Instrument-free argentometric determination of chloride via trapezoidal distance-based microfluidic paper devices.

Chloride (Cl-) is an inorganic anion present in a broad range of samples (e.g. biological, environmental, food, water, etc.), the determination of which is of widespread significance. In this work, we translate the well-established traditional argentometric method (Mohr's precipitation titration) into a small, simple, portable, and low-cost paper-based microfluidic diagnostic device, which provides rapid and quantitative analysis. The developed device enables the determination of chloride sample volumes as small as 5 µL. A distance-based detection method is implemented providing fully instrument-free quantitation. The beneficial effects of channel geometry (variable widths with constant heights) on analytical parameters were investigated. Trapezoidal channels (channel width changes linearly with height) were used to create a gradient of paper surface (titrant) available for the reaction, compared to the typical uniform rectangular channels (constant channel width). The trapezoid with increasing width offered higher sensitivity and lower detection limits (i.e. 0.05 mM vs 0.1 mM from the rectangular channel) for chloride determination across the concentration range of 0.05-25 mM. In addition, the effect of concentration of the deposited reagent on the obtained distance signals was investigated using varying concentrations of titrant (AgNO3), which allowed determination of chloride across a wider dynamic range (up to 200 mM). The utility of the paper devices was demonstrated by determination of chloride in a variety of matrices including body fluids (sweat, serum, and urine) and water samples (drinking, mineral, river water).

Prospects of pulsed amperometric detection in flow-based analytical systems - A review.

Electrochemical (EC) detection techniques in flow-based analytical systems such as flow injection analysis (FIA), capillary electrophoresis (CE), and liquid chromatography (LC) have attracted continuous interest over the last three decades, leading to significant advances in EC detection of a wide range of analytes in the liquid phase. In this context, the unique advantages of pulsed amperometric detection (PAD) in terms of high sensitivity and selectivity, and electrode cleaning through the application of pulsed potential for noble metal electrodes (e.g. Au, Pt), have established PAD as an important detection technique for a variety of electrochemically active compounds. PAD is especially valuable for analytes not detectable by ultraviolet (UV) photometric detection, such as organic aliphatic compounds and carbohydrates, especially when used with miniaturised capillary and chip-based separation methods. These applications have been accomplished through advances in PAD potential waveform design, as well as through the incorporation of nanomaterials (NMs) employed as microelectrodes in PAD. PAD allows on-line pulsed potential cleaning and coupling with capillary or standard separation techniques. The NMs are largely employed in microelectrodes to speed up mass and electron transfer between electrode surfaces and to perform as reactants in EC analysis. These advances in PAD have improved the sensitive and selective EC detection of analytes, especially in biological samples with complex sample matrices, and detection of electro-inactive compounds such as aliphatic organic compounds (i.e., formic acid, acetic acid, maleic acids, and ß-cyclodextrin complexes). This review addresses the fundamentals of PAD, the role of pulsed sequences in AD, the utilisation of different EC detectors for PAD, technological advancements in PAD waveforms, utilisation of microelectrodes in PAD techniques, advances in the use of NMs in PAD, the applications of PAD, and prospects for EC detection, with emphasis on PAD in flow-based systems.

High-throughput deposition of chemical reagents via pen-plotting technique for microfluidic paper-based analytical devices.

The deposition of chemical reagent inks on paper is a crucial step in the development and fabrication of microfluidic paper-based analytical devices (µPADs). A pen-plotting approach, delivering chemical ink deposition using technical pens filled with reagents and inserted into a desktop electronic plotter, is shown herein to be a versatile, low-cost, simple, rapid, reproducible, and high-throughput solution. The volume of the deposited ink was quantified gravimetrically, confirming that nanoliter volumes of reagents can be deposited reproducibly (e.g. 7.55 ±â€¯0.37 nL/mm for a plotting speed of 10 cm/s) in detection zones of µPADs, typically spatially defined using wax printing. This approach was further investigated with regard to deposition of reagents in different geometrical forms (circular and linear), so demonstrating its applicability for preparation of µPADs with flexible design and application. By adjusting the plotting speed for linear deposition, lines with a relatively large range of widths (≈628-1192 µm) were created. Circular deposition was also demonstrated via delivery of reagents within wax printed circular fluidic barriers of a range of diameters (inner diameter = 1.5-7 mm). These capabilities were practically demonstrated via the fabrication of µPADs, based upon differing detection principles for determination of aluminum in natural waters using Morin as the fluorescent reagent. Traditional µPADs based on digital image colorimetry (DIC) were produced using circular deposition, whilst distance-based µPADs exploited linear deposition. Both types of µPADs developed using this method showed excellent precision for determination of trace concentrations of aluminium (average RSDs = 3.38% and 6.45%, and LODs = 0.5 ng (0.25 ppm) and 2 ng (0.5 ppm), for traditional and distance-based detection, respectively).