Plastic surface-immobilized primers for integrated DNA capture and purification in simplified molecular diagnostics - a proof-of-concept study

Current molecular diagnostic (MDx) platforms rely on silica-coated surfaces for nucleic acid extraction. This requires multiple wash buffers and complex automation, thereby creating cost and usability barriers for resource-limited settings. Several modifications of MDx towards affordability adopt miniaturization technologies such as microfluidics, underscoring the potential impact of material reduction on MDx cost. Most advancements, however, replicate the same silica-based process steps leaving its cost-implication unaddressed. This study positions oligonucleotide-functionalized surfaces as a potential alternative approach for DNA capture with the potential for future integration into lab-on-a-chip (LoC) technologies. Hybridization-based genomic DNA and oligo capture were first validated using oligonucleotide-functionalized microparticles as a surrogate for polycarbonate plastic surfaces. Polycarbonate plastic was selected due to its biocompatibility, thermal tolerance, and the possibility of primer immobilization through amide bond. To enable the immobilization of a high concentration of oligonucleotides on polycarbonate plastics, their tubes were sequentially treated through acetone splash (10 s exposure) and UV photo-oxidation (10 min) to increase their carboxyl density. Combined acetone and UV treatments increased the tubes’ carboxyl density 2- fold (2.02 × 10 –3 to 4.0 × 10 –3 nmol/mm 2). To further improve the primer-carrying capacity of the tube surface, the oligonucleotide loading capacity of two linker molecules, branched polyethyleneimine (BPEI) and ethylenediamine (EA), was compared. BPEI linkers provided 2.6-fold higher oligonucleotide payload compared to ethylenediamine. The concentration of surface-immobilized oligonucleotides available for DNA capture was quantified fluorometrically from RFU values referenced against a calibration curve. An average of 0.125 ± 0.018 μ M and 0.0296 ± 0.0132 μ M oligonucleotides were immobilized in a 25 μ L working volume using BPEI and EA respectively. Although the concentration achieved using BPEI is theoretically compatible with 2-primer amplification chemistries such as PCR and recombinase polymerase amplification (RPA), further optimization is required to enable LAMP. Optimization efforts addressing the tubes’ tapered geometry and the need for mixing during functionalization is recommended to achieve higher primer concentrations. This proof-of-concept establishes preliminary technical feasibility of primer-functionalized surfaces as a potential alternative to silica-based nucleic acid extraction, pending full workflow integration. The validated surface engineering approach provides a development pathway for a cost-effective diagnostic platforms suitable for resource-limited settings.

Sub-Saharan Africa (SSA) bears the highest global infectious disease burden and remains a key region for clinical testing of emerging diagnostics, yet many advanced molecular technologies remain inaccessible because of high cost (National Institute of Allergy and Infectious Diseases, 2024; Reiner et al., 2022; Gouda et al., 2019; Ouma Mugabe et al., 2020; People’s Vaccine Alliance, 2023). Molecular diagnostics (MDx) exemplify this challenge, remaining expensive and unaffordable in the region without subsidization. The high cost of MDx platforms may be partly due to a compensatory design strategy. A compensatory design strategy refers to a complex assembly of materials and processes used to correct or offset inherent limitations of a core design choice, rather than eliminating the limitation itself. This design framework consequently inflates the cost and complexity of the product or process being designed. In state-of-the-art MDx platforms, this compensatory design strategy is exemplified by the need for multiple wash buffers, elution buffer, and magnets for nucleic acid extraction. These additional materials and process steps are compensatory consequences of the choice of silica-coated surfaces as the nucleic acid capture medium.

This study identifies design-related cost-drivers in current molecular diagnostic systems and proposes an alternative solid-phase DNA-capture approach using primer-functionalized plastic surfaces.

Some design inefficiencies of most state-of-the-art point-of-care molecular diagnostic platforms

MDx platforms such as the Cepheid GeneXpert, Biofire FilmArray, and Abbott IDNOW adopt Boom chemistry (Boom et al., 1990) - a widely used sample preparation technique for nucleic acid extraction and purification. In Boom chemistry, illustrated in Figure 1A, silica-coated surfaces, especially paramagnetic particles, are used to non-specifically bind nucleic acid from the sample suspension to enable their isolation and further handling. Although effective, this method presents significant drawbacks: High material cost: This is due to the need for multiple reagents, process steps and materials. Additionally, adoption of process automation as exemplified by (Derfus et al., 2024; Quitoviera et al., 2022; Ohashi et al., 2020; Fang et al., 2007) for particle handling significantly increases instrument cost and complexity.

Significant Target Loss: The steps associated with Boom chemistry have been reported to result in a ∼ 45%–50% loss of target NA (Katevatis et al., 2017; Bordelon et al., 2013). This often impacts assay sensitivity, especially in low analyte titre samples.

Risk of Inhibition: Due to the non-specificity of the nucleic acid capture step, non-target nucleic acid (for example, human genomic DNA) competes with the target biomarkers for the microparticle surface, leading to inhibition (Latham et al., 2023) or poor sensitivity. In addition, nonspecific interactions between particles and other biomolecules, such as lipoproteins, can form biocorona (Lima et al., 2020), thereby limiting the available surface area for nucleic acid capture.

Logistical Challenges: The required alcohol wash buffers present supply chain and logistical challenges due to their high ethanol concentration which requires specialized storage conditions for shipping. Although most nucleic acid extraction kits manufacturers expect the end-user to provide ethanol locally, there is an unacknowledged risk of supply of contaminated ethanol (Ogah et al., 2024), especially in SSA. Impurities in the wash buffers due to adulteration in the supplied ethanol pose the risk of performance variability.

Nucleic acid capture and purification

The choice of nucleic acid capture using silica-coated paramagnetic microparticles (Boom chemistry) prompts the need for well-to-well particle movement as simplified in Figure 1A (methods 1 & 2), alcohol wash steps, and target elution as compensatory steps. Thus, an alternative nucleic acid capture method that could eliminate all compensatory processes and material needs would save cost.

Since most molecular diagnostic test consumables and MDx-based lab-on-a-chip platforms are made of plastics, the surface properties of these plastics could be exploited for nucleic acid isolation and purification. As depicted in Figure 1B, hypothetically, cell-free nucleic acid can be captured using surface-immobilized primers. It is projected that exploiting the surface of plastic tubes may offer a pathway to reducing user/bulk instrument-driven process steps for nucleic acid extraction and purification from 10 to 3 (Figure 1A VS 1B), potentially representing ∼ 70% decrease in process steps. Should this reduction be realised, it is further anticipated that consumable part count might decrease proportionally from 10 (4x tubes, 4x buffers, 1x magnet, ×1 microparticle) to 3 (1x tube, 2x buffers).

Working theory

After cell lysis, plastic surface-immobilized primers recognize and bind to complementary nucleic acid targets. The tube is then washed with alcohol-free buffers to prevent carryover of impurities and lysis reagents such as guanidine. An alcohol-based wash buffer is not required because nucleic acid precipitation is not required. After the wash step, an amplification master mix can be added for target amplification with surface-immobilized primers (Figure 1B). Hence, the immobilized primers perform two functions: nucleic acid capture, and amplification.

The success of this design-thinking approach to affordable MDx, however, depends on: The feasibility of DNA-DNA hybridization in lysis conditions;

The feasibility of immobilizing primers on plastic surface; and

The possibility of immobilizing adequate concentration of primers on the plastic surface for loop-mediated isothermal amplification (LAMP).

This proof-of-concept study presents early validation data derisking the above dependencies for future advancement and adoption in LoC MDx platforms.

The following section outlines the surface-chemistry procedures and primer-immobilization strategies used to realize this simplified, solid-phase diagnostic approach.

Validation of solid-phase DNA capture during lysis

The feasibility and efficiency of DNA target capture using surface-immobilized oligonucleotides and primers was determined as a preliminary step via fluorescence resonance energy transfer, FRET. To achieve this, the fluorescence intensity of COOH-derivatized PMMA microparticles (PolyAn, GmbH) functionalized with Cy5-O (ref. Table 2) was visually compared before and after a hybridization protocol to Cy5-O FRET pair. The particles were functionalized using methods detailed by Anyaduba et al. (2026).

During hybridization, the mixture containing immobilized Cy5-O and quencher-labelled Cy5-O-complement was heated to 95 °C for 2 min and cooled to 40 °C) before imaging via confocal microscopy. Efficient DNA capture was qualitatively determined by the absence of ligand fluorescence around the particles after the hybridization process. Since the DNA capture process would occur during the lysis step, the hybridization protocol was evaluated in both lysis (Qiagen ATL) and phosphate buffers to determine the effect of chemical lysis on target capture. BPEI was used as a linker given its higher oligo-carrying capacity compared to EA. After the hybridization protocol, the microparticles were washed three times via vortexing using PBS to ensure removal of any adsorbed quencher-labeled Cy5-O-complement.

Validation of bacterial genomic DNA capture using surface-immobilized primers

In addition to demonstrating primer-target hybridization via FRET, the feasibility of capturing bacterial genomic DNA using surface-immobilized primers was validated. For this, E. coli genomic DNA was extracted from cells grown in LB broth using the QuickExtract DNA extraction kit (Bioresearch Technologies). After quantification, 10 μ L of the DNA extract containing ∼ 4.8 × 1 0 6 genomic DNA copies was mixed with Magnabind microparticles pre-functionalized with E. coli FIP primers as a surrogate to primer-functionalized plastic surfaces for conceptual proof (Schoepp et al., 2017). Before mixing the DNA aliquot with the microparticles, the suspension fluid of the microparticles was removed via pipetting. This setup was repeated for different amounts of functionalized microparticles (0, 2.5, 5, 7.5, and 10 μ L). The mixtures were pipette-mixed and incubated at 95 °C for 2 min at 1500 RPM and allowed to cool to 40 °C. After incubation, the microparticles were magnetically separated from the DNA suspension before loop-mediated isothermal amplification at a final 10-fold dilution.

Plastic selection and surface optimization

The molecular structure of various plastics was used to determine the most suitable material on which primers can be immobilized without significant surface treatments (Figure 2). Although polyethylene terephthalate (PET) has been widely adopted for similar applications (Li et al., 2005a; Hosseini et al., 2014; Li et al., 2005b), they often require polyacrylic acid grafts to generate carboxyl-terminated functional groups (-COOH). Polycarbonate was selected because it avoids the grafting step, is thermally stable and biocompatible for molecular applications, and contains innate terminal carbonate groups. Through surface amination, the terminal carbonate groups could be functionalized with primary amines (Zhong et al., 2012; VanDelinder et al., 2015) in 72 h. They can also be photo-oxidized to reveal -COOH terminal groups before amine functionalization within 2 h. The option of photo-oxidation as a rapid prototyping measure that is amenable to limited resource settings due to its cost-effectiveness.

Plastic surface optimization: COOH density determination

It is expected that a high primer loading would lead to improved DNA capture efficiency. Consequently, to determine the amount of primers that can be immobilized for DNA capture on the plastic surface, the density of carboxyl functional groups (-COOH) on the tube was first determined. Since each COOH moiety can ideally be conjugated to one amine-terminated biomolecule, the -COOH density was used as a surrogate to determine the primer-carrying capacity of the tube surface. Hence, toluidine blue O dye (TBO) assay, which assumes 1:1 ratio of carboxyl group to TBO dye molecules was adopted (Li et al., 2005a; Hosseini et al., 2014; Li et al., 2005b). For this analysis, polycarbonate tubes (Costar ModelP, Corning Thermowell) were filled with 200 μ L 0.5 mM TBO (Sigma Aldrich) reconstituted in 0.1 mM NaOH and incubated ambiently for 10 h (long incubation) or 2 h (short incubation). After incubation, the tubes were washed copiously and incubated ambiently for 10 min with 0.1 mM NaOH to remove any adsorbed dye. After the wash steps, carboxyl-bound TBO dye molecules were eluted with 200 μ L 50% (v/v) acetic acid. The absorbance of the eluted dye was measured via spectrophotometry (FLUOstar Omega) at 630 nm (Jackson et al., 2014). The concentration of the eluted dye was determined from a calibration curve.

COOH density optimization via surface abrasion

To optimize the COOH density of the polycarbonate tube surface, there was a need to increase the available surface area. Hence, the tubes were subjected to various treatment regimens to increase surface area. These regimens include: 10 s splash with acetone, 30 min exposure to acetone vapour at room temperature, 5 and 10 min exposure to acetone vapour at 60 °C. Additionally, they were exposed to a 5-min irradiation process with UV (245 nm UV, Hoefer UVC 500 Ultraviolet crosslinker). Acetone treatment was evaluated due to its effect on surface area (Brown and Bhushan, 2016), while UV was attempted on the basis of its ability to disintegrate the molecular structure of polycarbonate thereby exposing more -COOH functional groups (Li et al., 2005b; Bae et al., 2022). The effect of combining both UV and acetone treatment methods, as well as their treatment order was also evaluated. Temporal analysis of UV exposure from 2 to 15 min on pristine and acetone-treated tubes was also conducted.

Design and validation of primer payload models for DNA capture on plastic tube surface

To optimize primer delivery (payload) for DNA capture, we developed two primer payload models primarily differentiated by the choice of linker molecules (Figure 5A).

Model 1 - 1:1: x COOH:linker:primer model

(x ≤ 1) A 1:1: x COOH:Linker:Primer involves the covalent immobilization of primers to the surface of plastics such that each COOH molecule can carry only one primer molecule using unbranched linker molecules. The presence of two terminal primary amine groups on ethylenediamine (EA) makes it an ideal linker for 1:1 crosslinking of the primers to the carboxyl groups on the tube. To validate this, carboxyl-derivatized paramagnetic microparticles (Magnabind, ThermoScientific) was used. The surfaces were functionalized following 1-Ethyl-3-diaminopropyl carbodiimide (EDC) protocols in which 20 mM EA was used as a linker molecule.

Model 2 - 1: n: x COOH:linker:primer model (1 ≤ n < ∞, 0 ≤ x ≤ 1)

In this model, each available COOH moiety is functionalized to carry x primer molecules on each of the N amine groups of the branched linker molecules. Model 2 was necessitated by the limited surface area available for primer immobilization in a tube at 25 μ L working volume. Relative to the fluid height, h, and the tube geometry, the surface area (Equation 1) available for primer immobilization using the tube (Costar ModelP, Corning Thermowell)is reduced from 170.99 mm 2 (H = 13.1 mm) to 73.60 mm 2.

Ideally, in model 2, dendrimeric polyamine molecules such as poly (amidoamine) and branched polyethyleneimine, BPEI can be used. Each immobilized dendrimeric polyamine molecule provides access to “ N ″ number of primary amine groups. Due to cost and ease of access, BPEI (M n = 10,000) was adopted as a linker. Thus, 0.3 g BPEI/mL of 0.1 M MES buffer was adopted as a linker.

Tube surface functionalization with linker molecules and oligonucleotides

The feasibility of covalently immobilizing BPEI onto the surface of the plastic was evaluated. This was conducted through a temporal evaluation of the amine coupling process at 5, 10, 20, 30, and 40 min using acetone and UV pre-treated tubes. TBO assay was then used to determine the mean percentage reduction in -COOH concentration as a surrogate for successful covalent immobilization of BPEI as well as its efficiency.

It is expected that using BPEI as a linker, in comparison to EA, will yield an N -fold increase in the amount of primers immobilized. Hence, the value of N was also investigated.

Evaluation of BPEI primer payload in comparison to EA (model 1 VS model 2)

Preliminary confirmation of higher primer payload capacity of the BPEI-modified surface was carried out using carboxyl-derivatized microparticles (Magnabind, ThermoFisher) linked to 5′ phosphate- and 3′ 6-FAM terminated oligonucleotide sequences (FAMO, - Table 1). First, the linker molecules (EA and BPEI) were covalently linked to the -COOH derivatized microparticles prior to FAMO immobilization via phosphoramidate linkage (Ligaj et al., 2006; Wickramathilaka and Tao, 2019) for FAMO-enabled flow cytometric comparison of the ligand intensity of EA and BPEI-functionalized microparticles. The difference in the amount of FAMO immobilized on the microparticles using the models was determined via Overton subtraction of their ligand fluorescence intensities. The comparative analysis was carried out using microparticle populations outlined in Table 2.

Evaluation of EA and BPEI-linked primer payload and DNA capture in polycarbonate tubes

The value of “ N ″ was calculated using Equation 2 as the ratio of ligand fluorescence due to BPEI to ligand fluorescence due to EA linkage.

Proof-of-concept: primer-target hybridization

Figure 3 provides evidence of the feasibility of capturing target nucleic acids using surface-immobilized complementary oligonucleotides on microparticle surrogates for plastic surfaces. As shown in Figure 3B, hybridization of the FRET pair led to the complete loss of ligand fluorescence on the microparticle surfaces. The complete loss of ligand fluorescence on the microparticles for both PBS and lysis buffer conditions provides evidence of the feasibility of DNA target capture in the presence of lysis buffer containing chaotropic salts, guanidinium thiocyanate.

Figure 3C shows a Logistic 4P-fit of each test condition (n = 3). The successive increase in the time-to-positive correlates with the increase in the amount of functionalized particles spiked for DNA capture. As expected, amplification efficiency decreases as the amount of free-DNA in the suspension decreases, hence the increase in standard deviation from ± 0.36 (no microparticles) to ± 5.03 (10 μ L microparticles), exemplifying the feasibility of using surface-immobilized primers to capture genomic DNA on the functionalized surrogate particles.

Tube surface preparation and surface area optimization

Primary functionalization: chemical pretreatment using acetone

The effect of treating the polycarbonate tubes with acetone was determined via TBO assay. As determined from the calibration curve on Figure 4B, exposing the tubes to acetone splash yielded a ∼ 25% increase in COOH density compared to pristine tubes Figures 4C,D).

The mean COOH density increased from 2.02 × 1 0 − 3 nmole/mm 2 (total TBO = 0.35 ± 0.01 nmole, n = 3; 2.33 μ M) for the pristine tubes to 2.5 × 1 0 − 3 nmole/mm 2 (total TBO = 0.43 ± 0.01 nmole, n = 3; 2.87 μ M) for acetone splash - Figure 4D.

While exposure to acetone vapour at 60 ° C for 10 min showed promise (Figures 4C,E), it required a controlled setup and instrumentation to achieve. Exposing the tubes to 60 ° C acetone vapour for any length of time also irreversibly deformed the tubes, making them difficult to handle. A dissimilar result was experienced on tubes exposed to acetone vapour at RT for 30 min, however, the tubes became more hydrophobic preventing contact of the dye solution with the tube walls thus yielding lower -COOH density (1.65 × 1 0 − 3 nmole/mm 2; total TBO = 0.28 ± 0.01 nmole, n = 3; 1.867 μ M) than the control group (Figure 4C). Further efforts to reduce the exposure time to acetone vapour at RT were not pursued because the process required a setup that would increase handling complexity. As shown in Figure 4D, the most effective treatment method was pipetting acetone into the tubes for 10 s (acetone splash). Prolonged incubation of the tubes beyond 15 s, however, resulted in the disintegration of the tubes. It is worthwhile to mention that the rate of disintegration was impacted by the thickness of the tubes as faster deterioration was experienced at the thinnest part of the tubes.

Secondary functionalization: optical pretreatment using UV

Using a shorter TBO incubation time of 2 h, exposing the tubes to UV showed a ∼ 22% increase in COOH density (9.12 × 1 0 − 4 nmole/mm 2, Total TBO = 0.156 ± 0.003 nmole, n = 3; 1.04 μ M) compared to the control (pristine) (7.44 × 1 0 − 4 nmole/mm 2, Total TBO = 0.127 ± 0.003 nmole, n = 3; 0.8467 μ M) Figure 4F. Increasing the UV exposure time of pristine tubes beyond 5 min did not yield statistically significant improvements (P Value: 0.8403; α = 0.05).

Pre-treating the tubes using UV and then acetone splash showed a linear increase in COOH yield until 10 min (R 2 = 0.922, slope = 0.15). Beyond 10 min UV incubation (15 min) the COOH density did not yield any improvements beyond 7.5 and 2-min incubation (Figure 4G). Further, in reference to Figure 4G, a pairwise Tukey-Kramer comparison of the effects of UV exposure for 7.5 and 10 min and then acetone showed no statistically significant difference (P Value = 0.4367; α = 0.05).

As deduced from Figure 4H, by combining acetone splash and UV pretreatments in the right order, a mean COOH density of 4.0 × 1 0 − 3 nmole/mm 2 (4.56 μ M) was achieved using a 10-h TBO incubation.

Considering that a combination of UV and acetone treatments yielded higher COOH density, the treatment order was evaluated. As shown in Figure 4H, primary treatment with acetone before a secondary treatment with UV yielded 15% increase in COOH density (P value: 0.018). As such, a repeat of the temporal studies comparing 7.5 min UV incubation against 10 min with acetone as the primary treatment was conducted. This showed a 9% increase in COOH density with a P value of 0.0486 (Figure 4I).

Ideally, DNA capture should occur at a fluid height equal to the height of the working volume of the amplification master mix. At a 25 - 30 μ L working volume, the effective surface area of the tube available for DNA capture reduces from ∼ 171 to ∼ 74 mm 2 (Figure 4A). Since the tube is tapered at the bottom, the COOH-density cannot be directly calculated from the reported data. Hence, the amount of primers that can be immobilized within this working volume was directly evaluated and optimized.

Determination of surface functionalisation efficiency following model 2

The primer payload of model 2 was evaluated using the tubes. For this, BPEI was covalently immobilized to the surface of the tubes to investigate the efficiency of oligo functionalization on the plastic surface. As shown in Figure 5B, functionalization of acetone and UV-pretreated tubes with BPEI for 10 min yielded a 56% reduction in TBO density on the tube surface. This translates to 56% efficiency of conjugating surface COOH groups to primary amine groups of BPEI. A temporal study to determine optimal incubation timing of BPEI (TBO incubation at 2 h) showed no significant difference (P value = 0.1618, ANOVA) for BPEI incubation from 5 min to 40 min (Figure 5C).

Comparison of models 1 and 2 on microparticle surfaces

To determine the value of “n”, the fluorescence intensity of two groups of microparticles functionalized with EA (model 1) and BPEI (model 2) and FAMO were compared via flow cytometry (Figures 5D,E). As shown in Figure 5F, for the same amount of microparticles (∼ 2.0 × 1 0 8 microparticles) and FAMO concentration in each microparticle group, only ∼ 50 % of the BPEI + microparticles were FAMO + compared to ∼ 93 % in EA+. This ∼ 43 % difference suggests that BPEI + surfaces conjugate more oligonucleotides per microparticle compared to EA + microparticles. By comparing the average ligand fluorescence intensity of the microparticles, “N” could be deduced as ∼ 2.61 (ratio of BPEI + microparticle ligand intensity to EA + microparticle ligand intensity) – Figure 5F.

Demonstration of hybridization-based oligonucleotide capture on primer-functionalized polycarbonate tube surfaces

Realistically, as deduced from Figure 5I, the value of N on the tubes was determined as ∼ 4.33. This was determined by taking the ratio of the mean concentration (μ M at 25 μ L working volume) of Cy5-O hybridized onto immobilized Cy5-O-complements on the surface of the tubes (n = 25). The concentration of hybridized Cy5-O was determined from the RFU values of the calibration curves Figures 5G,H.

An average of 0.125 ± 0.018 μ M and 0.0296 ± 0.0132 μ M Cy5-O concentration was hybridized onto Cy5-O-complement immobilized on the tubes’ surface (n = 25) with BPEI and EA, respectively (Figure 5I). While this recorded concentration stems from hybridized oligonucleotides, it provides an estimate of the concentration of surface-immobilized primers potentially accessible for DNA capture. While the inferred primer payload is not optimal for LAMP amplification (requires a total of 4.4 μ M primers), the achieved BPEI-linked concentration is theoretically ideal for 2-primer chemistries such as polymerase chain reaction (PCR) (Forootan et al., 2017), recombinase polymerase amplification (RPA) (TwistDx Ltd, 2019) and nicking enzyme amplification reaction (NEAR) (Ménová et al., 2013).

This evaluation was carried out using a working volume of 25 μ L without continuous mixing during incubation. Considering the tapered design of the tubes, at a working volume of 25–30 μ L, the low conjugation efficiency is expected due to the possibility of steric hindrance. To adapt this method with LAMP, there is a need for further surface area optimization via linearized surfaces, such as microfluidic channels.

This proof-of-concept study successfully demonstrates that primers can be immobilized on polycarbonate surfaces. Using carboxyl-derivatized particles as surrogate surfaces, it demonstrates that surface-bound primers can be used to capture genomic DNA and complementary oligonucleotides even in lysis conditions. The validated surface engineering approach establishes a technical foundation for primer immobilization on plastic surfaces.

Demonstrated technical achievements

The systematic surface optimization achieved significant improvements in functional capacity. Combined acetone treatment and UV photo-oxidation increased surface carboxyl group density. The branched polyethyleneimine (BPEI) linker strategy proved highly effective, delivering 2.6-fold higher oligonucleotide payload compared to linear ethylenediamine linkers, as confirmed through flow cytometry analysis.

Most importantly, FRET validation on microparticles demonstrated that surface-immobilized primers retain their hybridization functionality under chaotropic lysis conditions (a critical requirement for any integrated diagnostic workflow). The complete fluorescence quenching observed in both phosphate buffer and Qiagen ATL lysis buffer is consistent with the surface-based capture mechanism remaining functional in the presence of chaotropic agents typically encountered during MDx sample processing.

The achieved surface-bound primer concentration of 0.125 μ M at a 25 μ L working volume is theoretically compatible with 2-primer amplification chemistries such as PCR and recombinase polymerase amplification (RPA), which typically require 0.1-1.0 μ M per primer. This performance supports the potential of plastic surfaces as functional platforms for nucleic acid capture and amplification in simplified diagnostic workflows.

Development opportunities and scaling pathways

While the demonstrated primer concentration falls below the 4.4 μ M total required for loop-mediated isothermal amplification (LAMP), this gap identifies an addressable optimisation target for future surface area scaling strategies. To achieve target concentration, several complementary approaches could be pursued, including surface area enhancement via microfluidic geometries, improved conjugation efficiency beyond the current 56%, and exploration of higher-capacity primer payload models such as primer concatenation (discussed in a separate publication).

The demonstrated proof-of-concept with E. coli genomic DNA capture using surface-immobilized FIP primers, as demonstrated through the amplification experiments showing increased time-to-positive with increasing microparticle concentrations, provides encouraging evidence that the approach can transition from synthetic oligo hybridization to complex genomic targets.

The demonstrated surface chemistry relies entirely on standard laboratory reagents and common plastic substrates, creating a promising foundation for cost-effective advancement in resource-limited settings. The proposed approach has the potential to reduce dependence on magnetic particles, multiple wash buffers, and specialized handling equipment, which represent key barriers to diagnostic accessibility in low-resource environments. Future development will prioritize surface area optimization through linear channel or microfluidic designs, which could potentially achieve the 10–30 fold surface area increases necessary for complex amplification chemistries.

Research significance and future directions

This work establishes that fundamental process simplification, rather than incremental optimization of existing complex systems, represents a viable pathway toward globally accessible molecular diagnostics. By demonstrating preliminary evidence, through surrogate microparticles, that standard plastic consumables can be transformed into functional nucleic acid capture platforms, the study challenges conventional assumptions about the complex requirements for molecular diagnostic systems. The technical foundation validated here supports broader research directions in simplified diagnostic platform development. The demonstrated surface chemistry could be adapted to various plastic substrates and geometric configurations, potentially enabling the development of diverse diagnostic formats tailored to specific clinical needs and resource constraints. Additionally, surface COOH density was estimated using the TBO assay, an indirect colorimetric method widely employed for this purpose. Although the risk of nonspecific dye adsorption was mitigated through copious washing and prolonged incubation in NaOH, orthogonal surface characterisation techniques such as FTIR or XPS will provide direct chemical confirmation of surface functional groups. It, therefore, presents an avenue for strengthening surface characterisation in future work.

Study limitations

Treatment of the polycarbonate tubes was carried out to increase the surface area of the surface of the tubes, however, a direct measurement of the treatment impact on surface area was not directly quantified. The effect of dilute concentrations of acetone on the carboxyl-density was not included in the study. While the feasibility of genomic DNA capture using primer-functionalized microparticles as a surrogate for plastic surface was demonstrated, the study does not include specificity studies and the genomic DNA capture efficiency was not directly quantified. The FRET studies also follow the same concerns. No non-complementary oligonucleotide controls were included in the FRET experiment, which represents a limitation of the study. However, the distance-dependent nature of FRET provides indirect physical evidence of sequence specificity. FRET efficiency falls off proportionally to the 6th power of the gap between the pair (Forster equation). Thus, Energy transfer between the Cy5-labelled surface primer (donor) and the quencher-labelled complement (acceptor) sufficient to produce complete fluorescence quenching, as observed in Figure 3B, can only occur when the two molecules are within approximately 1–10 nm of each other (AAT Bioquest, 2026; Sun et al., 2013). This proximity is only achievable through sequence-specific Watson-Crick base pairing, which forces the donor and acceptor into a defined, nanometre-scale geometry. Nonspecifically adsorbed oligos would be randomly oriented and at variable distances from the surface-immobilized donor, making efficient FRET physically impossible. The complete and consistent fluorescence quenching observed across both PBS and lysis buffer conditions therefore constitutes direct physical evidence of sequence-specific hybridization, not nonspecific interaction. Additionally, the introduction of a wash step and the high salt conditions of the FRET experiments preclude non-specific adsorption on the microparticles or the linker molecules.

Furthermore, the tube functionalization experiments presented two major difficulties: inefficient mixing and geometric restrictions. The conical shape of the tubes impacts the available surface area for surface functionalization especially at 25 μ L working volume. Similarly, at a working volume of 25 μ L, mixing to enable controlled functionalization within the fluid height is near impossible without constrictive fluid manipulation. Hence, the suggested transition to a linearized fluid channel (microfluidics) for future explorations. Further advancements will also include a complete capture-wash-amplification workflow which was not demonstrated in this proof-of-concept study.

This proof-of-concept provides early evidence that simplified molecular diagnostic workflows, independent of complex automation, may be achievable through surface-immobilized primer chemistry. The validated principles suggest that diagnostic platforms prioritizing simplicity and accessibility represent an achievable goal for addressing global health diagnostic needs. The preliminary demonstration of DNA capture under lysis conditions using functionalized surfaces provides early experimental evidence to motivate further advancement of alternative DNA preparation technologies.