The detection module is composed of three different units for the following operations: mixing, gas diffusion and sensing. All three units were fabricated using the thermoplastic cyclic olefin co-polymer (COC). COC 8007 (of 25 μm thickness) and COC 6013 (of 400 μm thickness) were purchased from Tekni-Plex (Erembodegem, Belgium). The sensing unit included a reference electrode consisting of an Ag/AgCl paste (C2030812D3, Gwent, Pontypool, United Kingdom). It also included an ISE composed of a conductive support and an ion selective membrane. The former consisted of an epoxy-graphite resin constituted by 50% powdered graphite of 50 μm particle size (Merk, Germany), 36% epoxy Araldite-M (Ciba Geigy, Spain) and 14% hardener HR (Ciba Geigy, Spain). The latter was composed by 1% nonactin (Sigma-Aldrich, Spain), 33.5% PCV (Sigma-Aldrich, Spain) and 65.5% bis(1-butylpentyl)adipate (BBPA) (Sigma-Aldrich, Spain). Tetrahydrofuran (THF) (Sigma-Aldrich, Spain) was used as volatile solvent for the ion selective membrane.

To isolate NH4+ from the sample matrix, a hydrophobic polyvinylidene fluoride (PVDF) membrane of 125 μm thickness and 0.45 μm porous size purchased at Millipore was used. An additional membrane of polycarbonate (PC) with a porous size of 0.05 μm purchased at Whatman Nucleopore was used as a protective membrane to avoid direct contact between blood and the PVDF membrane.

Reagents used in this work included NH4Cl (Acros Organics, Belgium) for the preparation of the NH4+ stock solution, KCl (Sigma-Aldrich, Spain) to stabilize the potential at the reference electrode, NaOH (Sigma-Aldrich, Spain) and ethylenediaminetetraacetic acid (EDTA) (Panreac, Spain) as the donor solution and 2-[4-(2-hydroxyethyl)piperazin- 1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, Spain) as the acceptor buffer solution. Ba(OH)2 (Thermo Fisher Scientific, Spain) was used to adjust the pH of the buffer. All solutions were prepared using Milli-Q water. The control solution employed was the same as for the reference method (Ammonia Ultra Kit ABBOT, USA).

As mentioned before, the analytical system is composed of three different computer-controlled modules: fluid management, detection, and data acquisition and transmission (Fig. 1).

Schematic representation of the three modules that constitute the POC analytical system

The detection module consists of three different microfluidic units: (1) a micromixer where samples mix with a NaOH solution to transform NH4+ into volatile NH3; (2) a gas diffusion unit that contains the PVDF and the protective membrane; and (3) the sensing unit that includes the NH4+ ISE and the reference electrode. The design of the gas diffusion unit is one of the most critical parts of the detection module, and its original design from the previously published proof-of-concept has been modified to reduce pressure on the membranes surface and thus improving lifetime of the device.

The fluid management unit contains all the elements that are responsible for fluid handling, such as pumps, valves and control software. Data acquisition and data communication unit is composed by a potentiometer and a software that transfers data via Bluetooth, processes data and shows result on the display.

The compact POC dimensions (L x W x H) are 30 × 16 × 18 cm, and it weighs approximately 2 kg, which is adequate for its portability.

The fabrication technology of the different units that make up the detection module has been described in a previous publication [23]. The process is based on a layer-by-layer approach, alternating COC layers of different glass transition temperatures (Tg). This approach allows the use of the COC layer with a higher Tg as structural layers and those with a lower Tg as sealing layers. Patterns of structural layers are designed by a computer-aided design (CAD) software and transferred on top of the thermoplastic by micromilling using a computer numerically controlled (CNC) micromilling machine (Protomat C100/HF, LPKF, Spain). Then, the conductive support is placed and cured overnight at 40 °C followed by a polishing process. Afterwards, the ion selective membrane is drop-casted on the detection chamber over the conductive support. Finally, a thermo-compression press (Francisco Camps, Granollers, Spain) is used to seal all the COC layers together (T = 102 °C and P = 4 bar). Prior to use, the ISEs must undergo a conditioning step by submersion into a 0.1 M NH4Cl solution for 4 h and into a 10−4 M NH4Cl solution for 16 h.

The three different units of the detection module have been fabricated individually to facilitate the optimization of the analyzer and allow the replacement of one of the components, if necessary, as each unit has a different service lifetime. This cuts costs and reduces the time required for the fabrication. Real images of the three units can be seen in Fig. 2.

Real images of the different units that compose the detection module being: A the micromixing unit, B the sensing unit and C the gas diffusion unit in open position for membrane exchange

The fluid management unit includes a 4-channel peristaltic pump (Spetec, Germany), five 3-way injection valves (NResearch, NJ, USA) and a bubble trap (Elveflow, France). For the liquid propulsion, Tygon tubes of 1.14 mm and 0.64 mm internal diameter (Ismatec, Wertheim, Germany) and Teflon tubes of 0.8 mm internal diameter were used.

The signal acquisition and transmission module includes a custom made miniaturized potentiometer developed by the Research Centre for Biomedical Engineering (CREB) of Universitat Politècnica de Catalunya (UPC). Data transmission is via Bluetooth. A Labview program, also developed by the CREB, manages both modules: it controls all the fluid management elements of the system and handles signal acquisition and data processing. A schematic representation of the full setup can be seen in Fig. 3A, alongside real images of the top view (Fig. 3B) of the POC) and a screenshot of the developed Labview program (Fig. 3C).

A Schematic representation of the automated experimental setup, with the fluid management module (blue), detection module (green) and the data acquisition and transmission module (orange) and B real image of the top view of the POC device were 1: reference electrode, 2: indicator electrode, E: Potentiometer, T: bubble trap, Vx: 3-way injection valve, P: peristaltic pump, M: micromixer, G: gas diffusion unit, PC: computer, W: waste. C Screenshot of the interface of the developed Labview program, where there is the “start measurement” button to start the sample analysis, the ammonium concentration display to show the ammonium blood level, and there are other auxiliary buttons to stop the POC device and to perform an extra control analysis or calibration procedure, depending on the situation

The automated point-of-care (POC) system was installed in the HSJD laboratory for a 2-month continuous evaluation. During this period, 238 anonymized blood samples for routine ammonium determinations were analyzed concurrently using the novel POC system and the reference method. A 500 µL aliquot was collected anonymously, ensuring no sensitive patient information or identities were stored. The sampling batch included both spiked and non-spiked samples to cover the full expected physiological and pathological range of NH4+ in blood. All experiments were conducted within the HSJD laboratory, and no data was transferred to external hospital data repositories.

Blood samples were collected in tubes containing EDTA and analyzed in parallel. First, blood samples were analyzed by the POC; then, plasma fractions of these samples were analyzed by the reference method employed at the HSJD. For this purpose, plasma fractions were obtained after the centrifugation of blood samples for 10 min at 3000 rpm at 10 °C. The reference method consisted of an enzymatic method using an automated spectrophotometric procedure in an Architect ci8200 automated analyzer (ABBOT, Park, IL, USA) [8]. The reference method is accredited by ENAC agency following the ISO 15189 norm, and it is subjected to external and internal quality control schemes. These data are available on request.